HUMAN cDNAs AND PROTEINS AND USES THEREOF

ABSTRACT

The invention concerns GENSET polynucleotides and polypeptides. Such GENSET products may be used as reagents in forensic analyses, as chromosome markers, as tissue/cell/organelle-specific markers, in the production of expression vectors. In addition, they may be used in screening and diagnosis assays for abnormal GENSET expression and/or biological activity and for screening compounds that may be used in the treatment of GENSET-related disorders.

RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.10/485,231, filed Jul. 2, 2004, which is the national stage ofinternational application No. PCT/IB01/02321, filed Oct. 15, 2001, whichclaims priority from U.S. Provisional Application Ser. No. 60/311,305,filed Aug. 10, 2001; U.S. Provisional Application Ser. No. 60/314,734,filed Aug. 24, 2001; U.S. Provisional Application Ser. No. 60/318,204,filed Sep. 7, 2001; and U.S. Provisional Application Ser. No.60/326,470, filed Oct. 1, 2001 and entitled “Human cDNAs and Proteinsand Uses Thereof,” the disclosures of which are incorporated herein byreference in their entireties.

The Sequence Listing for this application is labeled “seq-list.txt”,which was created on May 4, 2007, and is 216 KB. The entire contents isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to GENSET polynucleotides andpolypeptides, and fragments, derivatives, and variants thereof. Thepresent invention also relates to recombinant vectors including thepolynucleotides of the present invention, particularly recombinantvectors comprising a GENSET gene regulatory region or a sequenceencoding a GENSET polypeptide, and to host cells containing thepolynucleotides of the invention. The invention further relates toantibodies that specifically bind to the polypeptides of the inventionand to methods for producing such antibodies and fragments thereof. Theinvention also provides for methods of detecting the presence of thepolynucleotides and polypeptides of the present invention in a sample,methods of diagnosis and screening of abnormal GENSET polypeptideexpression and/or biological activity, methods of screening compoundsfor their ability to modulate the activity or expression of the GENSETpolypeptides, and uses of such compounds.

BACKGROUND OF THE INVENTION

cDNAs encoding secreted proteins or fragments thereof represent aparticularly valuable source of therapeutic agents. Thus, there is aneed for the identification and characterization of secreted proteinsand the nucleic acids encoding them.

In addition to being therapeutically useful themselves, secretoryproteins include short peptides, called signal peptides, at their aminotermini which direct their secretion. These signal peptides are encodedby the signal sequences located at the 5′ ends of the coding sequencesof genes encoding secreted proteins. Because these signal peptides willdirect the extracellular secretion of any protein to which they areoperably linked, the signal sequences may be exploited to direct theefficient secretion of any protein by operably linking the signalsequences to a gene encoding the protein for which secretion is desired.In addition, fragments of the signal peptides calledmembrane-translocating sequences may also be used to direct theintracellular import of a peptide or protein of interest. This may provebeneficial in gene therapy strategies in which it is desired to delivera particular gene product to cells other than the cells in which it isproduced. Signal sequences encoding signal peptides also findapplication in simplifying protein purification techniques. In suchapplications, the extracellular secretion of the desired protein greatlyfacilitates purification by reducing the number of undesired proteinsfrom which the desired protein must be selected. Thus, there exists aneed to identify and characterize the 5′ fragments of the genes forsecretory proteins which encode signal peptides.

Sequences coding for secreted proteins may also find application astherapeutics or diagnostics. In particular, such sequences may be usedto determine whether an individual is likely to express a detectablephenotype, such as a disease, as a consequence of a mutation in thecoding sequence for a secreted protein. In instances where theindividual is at risk of suffering from a disease or other undesirablephenotype as a result of a mutation in such a coding sequence, theundesirable phenotype may be corrected by introducing a normal codingsequence using gene therapy. Alternatively, if the undesirable phenotyperesults from overexpression of the protein encoded by the codingsequence, expression of the protein may be reduced using antisense ortriple helix based strategies.

The secreted human polypeptides encoded by the coding sequences may alsobe used as therapeutics by administering them directly to an individualhaving a condition, such as a disease, resulting from a mutation in thesequence encoding the polypeptide. In such an instance, the conditioncan be cured or ameliorated by administering the polypeptide to theindividual.

In addition, the secreted human polypeptides or fragments thereof may beused to generate antibodies useful in determining the tissue type orspecies of origin of a biological sample. The antibodies may also beused to determine the cellular localization of the secreted humanpolypeptides or the cellular localization of polypeptides which havebeen fused to the human polypeptides. In addition, the antibodies mayalso be used in immunoaffinity chromatography techniques to isolate,purify, or enrich the human polypeptide or a target polypeptide whichhas been fused to the human polypeptide.

SUMMARY OF THE INVENTION

The present invention provides a purified or isolated polynucleotidecomprising, consisting of, or consisting essentially of a nucleotidesequence selected from the group consisting of: (a) the sequences of theodd SEQ ID NOs:1-23; (b) the sequences of clone inserts of the depositedclone pool; (c) the coding sequences of the odd SEQ ID NOs:1-23; (d) thecoding sequences of the clone inserts of the deposited clone pool; (e)the sequences encoding one of the polypeptides of the even SEQ IDNOs:2-24; (f) the sequences encoding one of the polypeptides encoded bythe clone inserts of the deposited clone pool; (g) the genomic sequencescoding for the GENSET polypeptides; (h) the 5′ transcriptionalregulatory regions of GENSET genes; (i) the 3′ transcriptionalregulatory regions of GENSET genes; (j) the polynucleotides comprisingthe nucleotide sequence of any combination of (g)-(i); (k) the variantpolynucleotides of any of the polynucleotides of (a)-(j); (l) thepolynucleotides comprising a nucleotide sequence of (a)-(k), wherein thepolynucleotide is single stranded, double stranded, or a portion issingle stranded and a portion is double stranded; (m) thepolynucleotides comprising a nucleotide sequence complementary to any ofthe single stranded polynucleotides of (l). The invention furtherprovides for fragments of the nucleic acids and polypeptides of (a)-(m)described above.

Further embodiments of the invention include purified or isolatedpolynucleotides that comprise, consist of, or consist essentially of anucleotide sequence at least 70% identical, more preferably at least75%, and even more preferably at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical, to any of the nucleotide sequences in (a)-(m)above, e.g. over a region of contiguous nucleotides at least about anyone integer between 10 and the last integer representing the lastinteger representing the last nucleotide of a specified sequence of thesequence listing, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide of the present inventionincluding (a) through (m) above.

The present invention also relates to recombinant vectors, which includethe purified or isolated polynucleotides of the present invention, andto host cells recombinant for the polynucleotides of the presentinvention, as well as to methods of making such vectors and host cells.The present invention further relates to the use of these recombinantvectors and recombinant host cells in the production of GENSETpolypeptides. The present invention further relates to a polynucleotideof the present invention operably linked to a regulatory sequenceincluding promoters, enhancers, etc.

The invention further provides a purified or isolated polypeptidecomprising, consisting of, or consisting essentially of an amino acidsequence selected from the group consisting of: (a) the full lengthpolypeptides of even SEQ ID NOs:2-24; (b) the full length polypeptidesencoded by the clone inserts of the deposited clone pool; (c) theepitope-bearing fragments of the polypeptides of even SEQ ID NOs:2-24;(d) the epitope-bearing fragments of the polypeptides encoded by theclone inserts contained in the deposited clone pool; (e) the domains ofthe polypeptides of even SEQ ID NOs:2-24; (f) the domains of thepolypeptides encoded by the clone inserts contained in the depositedclone pool; (g) the signal peptides of the polypeptides of even SEQ IDNOs:2-24 or encoded by the human cDNAs of the deposited clone pool; (h)the mature polypeptides of even SEQ ID NOs:2-24 or encoded by the humancDNAs of the deposited clone pool; and (i) the allelic variantpolypeptides of any of the polypeptides of (a)-(h). The inventionfurther provides for fragments of the polypeptides of (a)-(i) above,such as those having biological activity or comprising biologicallyfunctional domain(s).

The present invention further includes polypeptides with an amino acidsequence with at least 70% similarity, and more preferably at least 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similarity to thosepolypeptides described in (a)-(i), or fragments thereof, as well aspolypeptides having an amino acid sequence at least 70% identical, morepreferably at least 75% identical, and still more preferably 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to those polypeptidesdescribed in (a)-(i), or fragments thereof, e.g. over a region of aminoacids at least any one integer between 6 and the last integerrepresenting the last amino acid of a specified polypeptide sequence ofthe sequence listing. The invention further relates to methods of makingthe polypeptides of the present invention.

The present invention further relates to transgenic plants or animals,wherein said transgenic plant or animal is transgenic for apolynucleotide of the present invention and expresses a polypeptide ofthe present invention.

The invention further relates to antibodies that specifically bind toGENSET polypeptides of the present invention and fragments thereof aswell as to methods for producing such antibodies and fragments thereof.

The invention also provides kits, uses and methods for detecting GENSETgene expression and/or biological activity in a biological sample. Onesuch method involves assaying for the expression of a GENSETpolynucleotide in a biological sample using the polymerase chainreaction (PCR) to amplify and detect GENSET polynucleotides or Southernand Northern blot hybridization to detect GENSET genomic DNA, cDNA ormRNA. Alternatively, a method of detecting GENSET gene expression in atest sample can be accomplished using a compound which binds to a GENSETpolypeptide of the present invention or a portion of a GENSETpolypeptide.

The present invention also relates to diagnostic methods and uses ofGENSET polynucleotides and polypeptides for identifying individuals ornon-human animals having elevated or reduced levels of GENSET geneproducts, which individuals are likely to benefit from therapies tosuppress or enhance GENSET gene expression, respectively, and to methodsof identifying individuals or nonhuman animals at increased risk fordeveloping, or at present having, certain diseases/disorders associatedwith GENSET polypeptide expression or biological activity.

The present invention also relates to kits, uses and methods ofscreening compounds for their ability to modulate (e.g. increase orinhibit) the activity or expression of GENSET polypeptides includingcompounds that interact with GENSET gene regulatory sequences andcompounds that interact directly or indirectly with a GENSETpolypeptide. Uses of such compounds are also within the scope of thepresent invention.

The present invention also relates to pharmaceutical or physiologicallyacceptable compositions comprising, an active agent, the polypeptides,polynucleotides or antibodies of the present invention, as well as,typically, a physiologically acceptable carrier.

The present invention also relates to computer systems containing cDNAcodes and polypeptide codes of sequences of the invention and tocomputer-related methods of comparing sequences, identifying homology orfeatures using GENSET polypeptides or GENSET polynucleotide sequences ofthe invention.

In another aspect, the present invention provides an isolatedpolynucleotide, the polynucleotide comprising a nucleic acid sequenceencoding a polypeptide of the present invention including thepolypeptide of (a) through (i) above.

In another aspect, the present invention provides a non-human transgenicanimal comprising the host cell.

In another aspect, the present invention provides a method of making aGENSET polypeptide, the method comprising a) providing a population ofhost cells comprising a herein-described polynucleotide and b) culturingthe population of host cells under conditions conducive to theproduction of the polypeptide within said host cells.

In one embodiment, the method further comprises purifying thepolypeptide from the population of host cells.

In another aspect, the present invention provides a method of making aGENSET polypeptide, the method comprising a) providing a population ofcells comprising a polynucleotide encoding a herein-describedpolypeptide; b) culturing the population of cells under conditionsconducive to the production of the polypeptide within the cells; and c)purifying the polypeptide from the population of cells.

In another aspect, the present invention provides a biologically activepolypeptide encoded by any of the herein-described polynucleotides.

In one embodiment, the polypeptide is selectively recognized by anantibody raised against an antigenic polypeptide, or an antigenicfragment thereof, the antigenic polypeptide comprising any one of thesequences shown as even SEQ ID NOs:2-24 or any one of the sequences ofpolypeptides encoded by the human cDNAs of the deposited clone pool.

In another aspect, the present invention provides an antibody thatspecifically binds to any of the herein-described polypeptides andmethods of binding antibody to said polypeptide.

In another aspect, the present invention provides a method ofdetermining whether a GENSET gene is expressed within a mammal, themethod comprising the steps of: a) providing a biological sample fromsaid mammal; b) contacting said biological sample with either of: (i) apolynucleotide that hybridizes under stringent conditions to any of theherein-described polynucleotides; or (ii) a polypeptide thatspecifically binds to any of the herein-described polypeptides; and c)detecting the presence or absence of hybridization between thepolynucleotide and an RNA species within the sample, or the presence orabsence of binding of the polypeptide to a protein within the sample;wherein a detection of the hybridization or of the binding indicatesthat the GENSET gene is expressed within the mammal.

In one embodiment, the polynucleotide is a primer, and the hybridizationis detected by detecting the presence of an amplification productcomprising the sequence of the primer. In another embodiment, thepolypeptide is an antibody.

In another aspect, the present invention provides a method ofdetermining whether a mammal has an elevated or reduced level of GENSETgene expression, the method comprising the steps of: a) providing abiological sample from the mammal; and b) comparing the amount of any ofthe herein-described polypeptides, or of an RNA species encoding thepolypeptide, within the biological sample with a level detected in orexpected from a control sample; wherein an increased amount of thepolypeptide or the RNA species within the biological sample compared tothe level detected in or expected from the control sample indicates thatthe mammal has an elevated level of the GENSET gene expression, andwherein a decreased amount of the polypeptide or the RNA species withinthe biological sample compared to the level detected in or expected fromthe control sample indicates that the mammal has a reduced level of theGENSET gene expression.

In another aspect, the present invention provides a method ofidentifying a candidate modulator of a GENSET polypeptide, the methodcomprising: a) contacting any of the herein-described polypeptides witha test compound; and b) determining whether the compound specificallybinds to the polypeptide; wherein a detection that the compoundspecifically binds to the polypeptide indicates or inhibits or activatesof a specified biological activity that the compound is a candidatemodulator of the GENSET polypeptide.

BRIEF DESCRIPTION OF TABLES

Table I provides the Applicants' internal designation number (CloneID_Clone Name) which corresponds to each sequence identification number(SEQ ID NO) of the Sequence Listing, and indicates whether the sequenceis a nucleic acid sequence (DNA) or a polypeptide sequence (PRT).Further provided is information regarding the name of the correspondingnucleic acid or polypeptide sequence, and information regarding thedeposit of biological material. It should be appreciated that biologicalmaterials have been deposited with reference to their correspondingClone ID, Clone Name, or both Clone ID_Clone Name.

Table II provides the positions of the nucleotides of the correspondingSEQ ID NOs of the Sequence Listing which comprise the open reading frame(ORF), signal peptide, mature peptide, polyadenylation signal, and thepolyA tail of the polynucleotides of the invention.

Table III provides the positions of the amino acid of the correspondingSEQ ID NOs. of the Sequence Listing which comprise the positions ofimmunogenic epitopes of the polypeptides of the invention, which areuseful in antibody generation.

BRIEF DESCRIPTION OF SEQUENCES

Sequences are presented in the accompanying Sequence Listing.

Odd SEQ ID NOs:1-23 are the nucleotide sequences of cDNAs, with openreading frames as indicated. When appropriate, the potentialpolyadenylation site and polyadenylation signal are also indicated.

Even SEQ ID NOs:2-24 are the amino acid sequences of proteins encoded bythe cDNAs of odd SEQ ID NOs:1-23.

In accordance with the regulations relating to Sequence Listings, thefollowing codes have been used in the Sequence Listing to describesnucleotide sequences. The code “r” in the sequences indicates that thenucleotide may be a guanine or an adenine. The code “y” in the sequencesindicates that the nucleotide may be a thymine or a cytosine. The code“m” in the sequences indicates that the nucleotide may be an adenine ora cytosine. The code “k” in the sequences indicates that the nucleotidemay be a guanine or a thymine. The code “s” in the sequences indicatesthat the nucleotide may be a guanine or a cytosine. The code “w” in thesequences indicates that the nucleotide may be an adenine or a thymine.In addition, all instances of the symbol “n” in the nucleic acidsequences mean that the nucleotide can be adenine, guanine, cytosine orthymine.

In some instances, the polypeptide sequences in the Sequence Listingcontain the symbol “Xaa.” These “Xaa” symbols indicate either (1) aresidue which cannot be identified because of nucleotide sequenceambiguity or (2) a stop codon in the determined sequence whereapplicants believe one should not exist (if the sequence were determinedmore accurately). In some instances, several possible identities of theunknown amino acids may be suggested by the genetic code.

In the case of secreted proteins, it should be noted that, in accordancewith the regulations governing Sequence Listings, in the appendedSequence Listing the encoded protein (i.e. the protein containing thesignal peptide and the mature protein or fragment thereof) extends froman amino acid residue having a negative number through a positivelynumbered amino acid residue. Thus, the first amino acid of the matureprotein resulting from cleavage of the signal peptide is designated asamino acid number 1, and the first amino acid of the signal peptide isdesignated with the appropriate negative number.

In the case that a polynucleotide or polypeptide sequence described inthe specification for SEQ ID NOs:1-24 is in conflict with thecorresponding sequence provided in the Sequence listing, the sequencesprovided in the Sequence listing controls.

It should be appreciated that the polynucleotide and polypeptidesequences of SEQ ID NO:1-24 of the Sequence Listing are herebyincorporated by reference in their entireties.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTSDefinitions

Before describing the invention in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used to describe the invention herein.

The term “GENSET gene,” when used herein, encompasses genomic, mRNA andcDNA sequences encoding a GENSET polypeptide, including the 5′ and 3′untranslated regions of said sequences.

The term “GENSET polypeptide biological activity” or “GENSET biologicalactivity” is intended for polypeptides exhibiting any activity similar,but not necessarily identical, to an activity of a GENSET polypeptide ofthe invention. The GENSET polypeptide biological activity of a givenpolypeptide may be assessed using any suitable biological assay, anumber of which are known to those skilled in the art. In contrast, theterm “biological activity” refers to any activity that any polypeptidemay have.

The term “corresponding mRNA” refers to mRNA which was or can be atemplate for cDNA synthesis for producing a cDNA of the presentinvention.

The term “corresponding genomic DNA” refers to genomic DNA which encodesan mRNA of interest, e.g. corresponding to a cDNA of the invention,which genomic DNA includes the sequence of one of the strands of themRNA, in which thymidine residues in the sequence of the genomic DNA (orcDNA) are replaced by uracil residues in the mRNA.

The term “deposited clone pool” is used herein to refer to the pool ofclones deposited with the ATCC or other depositary authority.

The term “heterologous”, when used herein in reference to a polypeptideor polynucleotide, is intended to designate any polynucleotide orpolypeptide other than a particular GENSET polynucleotide or GENSETpolypeptide of the invention, respectively.

“Providing” with respect to, e.g. a biological sample, population ofcells, etc. indicates that the sample, population of cells, etc. issomehow used in a method or procedure. Significantly, “providing” abiological sample or population of cells does not require that thesample or cells are specifically isolated or obtained for the purposesof the invention, but can instead refer, for example, to the use of abiological sample obtained by another individual, for another purpose.

An “amplification product” refers to a product of any amplificationreaction, e.g. PCR, RT-PCR, LCR, etc.

A “modulator” of a protein or other compound refers to any agent thathas a functional effect on the protein, including physical binding tothe protein, alterations of the quantity or quality of expression of theprotein, altering any measurable or detectable activity, property, orbehavior of the protein, or in any way interacts with the protein orcompound.

“A test compound” can be any molecule that is evaluated for its abilityto modulate a protein or other compound.

An antibody or other compound that specifically binds to a polypeptideor polynucleotide of the invention is also said to “selectivelyrecognize” the polypeptide or polynucleotide.

The term “isolated” with respect to a molecule requires that themolecule be removed from its original environment (e.g., the naturalenvironment if it is naturally occurring). For example, anaturally-occurring polynucleotide or polypeptide present in a livinganimal is not isolated, but the same polynucleotide or polypeptide,separated from some or all of the coexisting materials in the naturalsystem, is isolated. Such polynucleotide could be part of a vectorand/or such polynucleotide or polypeptide could be part of acomposition, and still be isolated in that the vector or composition isnot part of its natural environment. Specifically excluded from thedefinition of “isolated” are: naturally-occurring chromosomes (such aschromosome spreads), artificial chromosome libraries, genomic libraries,and cDNA libraries that exist either as an in vitro nucleic acidpreparation or as a transfected/transformed host cell preparation,wherein the host cells are either an in vitro heterogeneous preparationor plated as a heterogeneous population of single colonies. Alsospecifically excluded are the above libraries wherein a specifiedpolynucleotide makes up less than 5% (may also be specified as 10%, 25%,50%, or 75%) of the number of nucleic acid inserts in the vectormolecules. Further specifically excluded are whole cell genomic DNA orwhole cell RNA preparations (including said whole cell preparationswhich are mechanically sheared or enzymatically digested). Furtherspecifically excluded are the above whole cell preparations as either anin vitro preparation or as a heterogeneous mixture separated byelectrophoresis (including blot transfers of the same) wherein thepolynucleotide of the invention has not further been separated from theheterologous polynucleotides in the electrophoresis medium (e.g.,further separating by excising a single band from a heterogeneous bandpopulation in an agarose gel or nylon blot).

The term “purified” does not require absolute purity; rather, it isintended as a relative definition. Purification of starting material ornatural material to at least one order of magnitude, preferably two orthree orders, and more preferably four or five orders of magnitude isexpressly contemplated. The term “purified” is further used herein todescribe a polypeptide or polynucleotide of the invention which has beenseparated from other compounds including, but not limited to,polypeptides or polynucleotides, carbohydrates, lipids, etc. The term“purified” may be used to specify the separation of monomericpolypeptides of the invention from oligomeric forms such as homo- orhetero-dimers, trimers, etc. The term “purified” may also be used tospecify the separation of covalently closed (i.e. circular)polynucleotides from linear polynucleotides. A substantially purepolypeptide or polynucleotide typically comprises about 50%, preferably60 to 90% weight/weight of a polypeptide or polynucleotide sample,respectively, more usually about 95%, and preferably is over about 99%pure but, may be specified as any integer of percent between 50 and 100.Polypeptide and polynucleotide purity, or homogeneity, is indicated by anumber of means well known in the art, such as agarose or polyacrylamidegel electrophoresis of a sample, or using HPLC. As an alternativeembodiment, purification of the polypeptides and polynucleotides of thepresent invention may be expressed as “at least” a percent purityrelative to heterologous polypeptides and polynucleotides (DNA, RNA orboth). As a preferred embodiment, the polypeptides and polynucleotidesof the present invention are at least; 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 96%, 96%, 98%, 99%, or 100% pure relative toheterologous polypeptides and polynucleotides, respectively. As afurther preferred embodiment the polypeptides and polynucleotides have apurity ranging from any number, to the thousandth position, between 90%and 100% (e.g., a polypeptide or polynucleotide at least 99.995% pure)relative to either heterologous polypeptides or polynucleotides,respectively, or as a weight/weight ratio relative to all compounds andmolecules other than those existing in the carrier. Each numberrepresenting a percent purity, to the thousandth position, may beclaimed as individual species of purity.

As used interchangeably herein, the terms “nucleic acid molecule(s)”,“oligonucleotide(s)”, and “polynucleotide(s)” include RNA or DNA (eithersingle or double stranded, coding, complementary or antisense), orRNA/DNA hybrid sequences of more than one nucleotide in either singlechain or duplex form (although each of the above species may beparticularly specified). The term “nucleotide” is used herein as anadjective to describe molecules comprising RNA, DNA, or RNA/DNA hybridsequences of any length in single-stranded or duplex form. Moreprecisely, the expression “nucleotide sequence” encompasses the nucleicmaterial itself and is thus not restricted to the sequence information(i.e. the succession of letters chosen among the four base letters) thatbiochemically characterizes a specific DNA or RNA molecule. The term“nucleotide” is also used herein as a noun to refer to individualnucleotides or varieties of nucleotides, meaning a molecule, orindividual unit in a larger nucleic acid molecule, comprising a purineor pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphategroup, or phosphodiester linkage in the case of nucleotides within anoligonucleotide or polynucleotide. The term “nucleotide” is also usedherein to encompass “modified nucleotides” which comprise at least onemodification such as (a) an alternative linking group, (b) an analogousform of purine, (c) an analogous form of pyrimidine, or (d) an analogoussugar (see, e.g., WO 95/04064). Preferred modifications of the presentinvention include, but are not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v)ybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, and2,6-diaminopurine. The polynucleotide sequences of the invention may beprepared by any known method, including synthetic, recombinant, ex vivogeneration, or a combination thereof, as well as utilizing anypurification methods known in the art (for teachings regarding thepreparation of modified oligos and nucleotides, see, e.g., U.S. Pat.Nos. 5,378,825; 5,386,023; 5,489,677; 5,602,240; and 5,610,289, U.S.Pat. Nos. 5,264,562 and 5,264,564, U.S. Pat. No. 5,223,618, U.S. Pat.No. 5,508,270, U.S. Pat. No. 4,469,863, U.S. Pat. No. 5,610,289 or5,625,050, U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, PCTapplications WO 94/17093 and WO 94/02499, U.S. Pat. No. 5,476,925, U.S.Pat. No. 5,023,243, U.S. Pat. Nos. 5,130,302 and 5,177,198).

The term “upstream” is used herein to refer to a location which istoward the 5′ end of the polynucleotide from a specific reference point.

The terms “complementary” or “complement thereof” are used herein torefer to the sequences of polynucleotides which is capable of formingWatson & Crick base pairing with another specified polynucleotidethroughout the entirety of the complementary region. For the purpose ofthe present invention, a first polynucleotide is deemed to becomplementary to a second polynucleotide when each base in the firstpolynucleotide is paired with its complementary base. Complementarybases are, generally, A and T (or A and U), or C and G. “Complement” isused herein as a synonym from “complementary polynucleotide”,“complementary nucleic acid” and “complementary nucleotide sequence”.These terms are applied to pairs of polynucleotides based solely upontheir sequences and not any particular set of conditions under which thetwo polynucleotides would actually bind. Unless otherwise stated, allcomplementary polynucleotides are fully complementary on the wholelength of the considered polynucleotide.

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymer of amino acids without regard to the length of thepolymer; thus, peptides, oligopeptides, and proteins are included withinthe definition of polypeptide. This term also does not specify orexclude chemical or post-expression modifications of the polypeptides ofthe invention, although chemical or post-expression modifications ofthese polypeptides may be included or excluded as specific embodiments.Therefore, for example, modifications to polypeptides that include thecovalent attachment of glycosyl groups, acetyl groups, phosphate groups,lipid groups and the like are expressly encompassed by the termpolypeptide. Further, polypeptides with these modifications may bespecified as individual species to be included or excluded from thepresent invention. The natural or other chemical modifications, such asthose listed in examples above can occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini, and may be present in the same or varying degreesat several sites in a given polypeptide. Also, a given polypeptide maycontain many types of modifications. Polypeptides may be branched, forexample, as a result of ubiquitination, and they may be cyclic, with orwithout branching. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcysteine, formation of pyroglutamate, formylation, gamma-carboxylation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, pegylation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination (see, for instance Creighton,(1993), Posttranslational Covalent Modification of Proteins, W.H.Freeman and Company, New York B. C. Johnson, Ed., Academic Press, NewYork 1-12; Seifter, et al., (1990) Meth Enzymol 182:626-646; Rattan etal., (1992) Ann NY Acad Sci 663:48-62). Also included within thedefinition are polypeptides which contain one or more analogs of anamino acid (including, for example, non-naturally occurring amino acids,amino acids which only occur naturally in an unrelated biologicalsystem, modified amino acids from mammalian systems, etc.), polypeptideswith substituted linkages, as well as other modifications known in theart, both naturally occurring and non-naturally occurring.

As used herein, the terms “recombinant polynucleotide” and“polynucleotide construct” are used interchangeably to refer to linearor circular, purified or isolated polynucleotides that have beenartificially designed and which comprise at least two nucleotidesequences that are not found as contiguous nucleotide sequences in theirinitial natural environment. In particular, these terms mean that thepolynucleotide or cDNA is adjacent to “backbone” nucleic acid to whichit is not adjacent in its natural environment. Additionally, to be“enriched” the cDNAs will represent 5% or more of the number of nucleicacid inserts in a population of nucleic acid backbone molecules.Backbone molecules according to the present invention include nucleicacids such as expression vectors, self-replicating nucleic acids,viruses, integrating nucleic acids, and other vectors or nucleic acidsused to maintain or manipulate a nucleic acid insert of interest.Preferably, the enriched cDNAs represent 15% or more of the number ofnucleic acid inserts in the population of recombinant backbonemolecules. More preferably, the enriched cDNAs represent 50% or more ofthe number of nucleic acid inserts in the population of recombinantbackbone molecules. In a highly preferred embodiment, the enriched cDNAsrepresent 90% or more (including any number between 90 and 100%, to thethousandth position, e.g., 99.5%) of the number of nucleic acid insertsin the population of recombinant backbone molecules.

The term “recombinant polypeptide” is used herein to refer topolypeptides that have been artificially designed and which comprise atleast two polypeptide sequences that are not found as contiguouspolypeptide sequences in their initial natural environment, or to referto polypeptides which have been expressed from a recombinantpolynucleotide.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A sequence whichis “operably linked” to a regulatory sequence such as a promoter meansthat said regulatory element is in the correct location and orientationin relation to the nucleic acid to control RNA polymerase initiation andexpression of the nucleic acid of interest. For instance, a promoter orenhancer is operably linked to a coding sequence if it affects thetranscription of the coding sequence.

The term “domain” refers to an amino acid fragment with specificbiological properties. This term encompasses all known structural andlinear biological motifs. Examples of such motifs include but are notlimited to leucine zippers, helix-turn-helix motifs, glycosylationsites, ubiquitination sites, alpha helices, and beta sheets, signalpeptides which direct the secretion of proteins, sites forpost-translational modification, enzymatic active sites, substratebinding sites, and enzymatic cleavage sites.

Although each of these terms has a distinct meaning, the terms“comprising”, “consisting of” and “consisting essentially of” may beinterchanged for one another throughout the instant application. Theterm “having” has the same meaning as “comprising” and may be replacedwith either the term “consisting of” or “consisting essentially of”.

Unless otherwise specified in the application, nucleotides and aminoacids of polynucleotides and polypeptides, respectively, of the presentinvention are contiguous and not interrupted by heterologous sequences.

As used herein, the term “tumor” refers to an abnormal mass orpopulation of cells that result from excessive cell division, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues. A “tumor” is further defined as two or more physicallyassociated neoplastic cells. The term “transformed cells,” “malignantcells” or “cancer” are interchangeable and refer to cells that haveundergone malignant transformation, and also includes lymphocytes thathave undergone blast transformation. Transformed cells have a greaterability to cause tumors when injected into animals, can typicallyproliferate without requiring adhesion to a substratum, and also lackcontact inhibition. The term “cancer” or “neoplastic disease”encompasses any type of cancer, in any tissue, and includes, but is notlimited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias.

The terms “inducing” or “inducing” with respect to a cellular process,e.g., apoptosis, refers to increasing or decreasing the number of cellsthat undergo the process, or the rate by which cells undergo theprocess, in a given cell population. Preferably the increase or decreaseis at least 1.25, 1.5, 2, 5, 10, 50, 100, 500 or 1000 fold increase ordecrease as compared to normal, untreated or negative control cells.

A “therapeutically effective amount”, in reference to the treatment of adisease or condition, refers to an amount of a compound that is capableof having any detectable, positive effect on any symptom, aspect, orcharacteristics of the disease or condition.

The terms “killing” or “inducing cytotoxicity” as used herein refer toinducing cell death by either apoptosis and/or necrosis, wherebyembodiments of the invention include only apoptosis, only necrosis andboth apoptosis and necrosis.

The terms “preventing” and “suppressing” as used herein refer toadministering a compound prior to the onset of clinical symptoms of adisease or condition so as to prevent a physical manifestation of thedisease or condition. The term “prophylaxis” is distinct from“treatment” and encompasses “preventing” and “suppressing”. Herein,“protection” includes “prophylaxis”. Protection need not be absolute tobe useful.

The term “treating” as used herein refers to administering a compoundafter the onset of clinical symptoms. The term “in need of treatment” asused herein refers to a judgment made by a caregiver that an individualor animal requires or will benefit from treatment. This judgment is madebased on a variety of factors that are in the realm of a caregiver'sexpertise, but that include the knowledge that the individual or animalis ill, or will be ill, as the result of a condition that is treatableby a compound of the invention.

The term “individual” or “patient” as used herein refers to any animal,including mammals, preferably mice, rats, other rodents, rabbits, dogs,cats, swine, cattle, sheep, horses, or primates, and most preferablyhumans. The term may specify male or female or both, or exclude male orfemale.

As used herein, the term “non-human animal” refers to any non-humananimal, including insects, birds, rodents and more usually mammals.Preferred non-human animals include: primates; farm animals such asswine, goats, sheep, donkeys, cattle, horses, chickens, rabbits; androdents, preferably rats or mice. As used herein, the term “animal” isused to refer to any species in the animal kingdom, preferablyvertebrates, including birds and fish, and more preferably a mammal.Both the terms “animal” and “mammal” expressly embrace human subjectsunless preceded with the term “non-human”.

As used herein, the terms “physiologically acceptable,”“pharmaceutically acceptable,” and “pharmaceutical” are interchangeable.

Identity Between Nucleic Acids or Polypeptides

The terms “percentage of sequence identity” and “percentage homology”are used interchangeably herein to refer to comparisons amongpolynucleotides and polypeptides, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Identity or similarity is evaluated using any of the varietyof sequence comparison algorithms and programs known in the art. Suchalgorithms and programs include, but are by no means limited to,TBLASTN, BLASTP, FASTA, TFASTA, CLUSTALW, FASTDB (Pearson and Lipman,(1988), PNAS 85(8):2444-2448; Altschul et al., (1990), J. Mol. Biol.215(3):403-410; Thompson et al. (1994), Nucleic Acids Res.22(2):4673-4680; Higgins et al., (1996), Meth. Enzymol. 266:383-402;Altschul et al., (1993), Nature Genetics 3:266-272; Brutlag et al.(1990) Comp. App. Biosci. 6:237-24).

In a particularly preferred embodiment, protein and nucleic acidsequence identities are evaluated using the Basic Local Alignment SearchTool (“BLAST”) which is well known in the art (e.g., Karlin andAltschul, (1990), PNAS 87:2267-2268; Altschul et al., (1997), Nuc. AcidsRes. 25:3389-3402). In particular, five specific BLAST programs are usedto perform the following task:

-   -   (1) BLASTP and BLAST3 compare an amino acid query sequence        against a protein sequence database;    -   (2) BLASTN compares a nucleotide query sequence against a        nucleotide sequence database;    -   (3) BLASTX compares the six-frame conceptual translation        products of a query nucleotide sequence (both strands) against a        protein sequence database;    -   (4) BLASTN compares a query protein sequence against a        nucleotide sequence database translated in all six reading        frames (both strands); and    -   (5) BLASTX compares the six-frame translations of a nucleotide        query sequence against the six-frame translations of a        nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis preferably obtained from a protein or nucleic acid sequence database.High-scoring segment pairs are preferably identified (i.e., aligned) bymeans of a scoring matrix, many of which are known in the art.Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet etal., (1992), Science 256:1443-1445; Henikoff and Henikoff, (1993),Proteins 17:49-61). Less preferably, the PAM or PAM250 matrices may alsobe used (see, e.g., Schwartz and Dayhoff, (1978), eds., Matrices forDetecting Distance Relationships: Atlas of Protein Sequence andStructure, Washington: National Biomedical Research Foundation). TheBLAST programs evaluate the statistical significance of all high-scoringsegment pairs identified, and preferably selects those segments whichsatisfy a user-specified threshold of significance, such as auser-specified percent homology. Preferably, the statisticalsignificance of a high-scoring segment pair is evaluated using thestatistical significance formula of Karlin (see, e.g., Karlin andAltschul, 1990). The BLAST programs may be used with the defaultparameters or with modified parameters provided by the user.

Another preferred method for determining the best overall match betweena query nucleotide or amino acid sequence (a sequence of the presentinvention) and a subject sequence, also referred to as a global sequencealignment, can be determined using the FASTDB computer program based onthe algorithm of Brutlag et al. (1990). In a sequence alignment thequery and subject sequences are both DNA or amino acid sequences. An RNAsequence can also be compared by first converting U's to T's. The resultof said global sequence alignment is in percent identity. Preferredparameters used in a FASTDB alignment of DNA sequences to calculatepercent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1,Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, GapPenalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of thesubject nucleotide sequence, whichever is shorter. Preferred parametersused in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2,Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0,Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter. If the subject sequence is shorter thanthe query sequence because of 5′ or 3′ deletions (for nucleotidesequences) or N- or C-terminal deletions (for amino acid sequences), notbecause of internal deletions, a manual correction must be made to theresults, because the FASTDB program does not account for terminaltruncations of a subject sequence when calculating percent identity. Themanual correction involves determining the percent of the total querysequence that is not aligned because of a truncation in the subjectsequence, and subtracting this percentage from the percent identitycalculated by the FASTDB program. This corrected score is what is usedfor the purposes of the present invention. No other manual correctionsare made for the purposes of the present invention.

Polynucleotides of the Invention

Many of the methods described in the present specification rely on theuse of common molecular biological techniques. A number of suchtechniques are taught, generally, in “Molecular Cloning; A LaboratoryManual”, 2d ed., Cole Spring Harbor Laboratory Press, Sambrook, et al.,eds., 1989, and “Methods in Enzymology; Guide to Molecular CloningTechniques”, Academic Press, Berger and Kimmel eds., 1987.

The present invention concerns GENSET genomic and cDNA sequences. Thepresent invention encompasses GENSET genes, polynucleotides comprisingGENSET genomic and cDNA sequences, as well as fragments and variantsthereof. These polynucleotides may be purified, isolated, orrecombinant.

Also encompassed by the present invention are allelic variants,orthologs, splice variants, and/or species homologues of the GENSETgenes. Procedures known in the art can be used to obtain full-lengthgenes and cDNAs, allelic variants, splice variants, full-length codingportions, orthologs, and/or species homologues of genes and cDNAscorresponding to a nucleotide sequence selected from the groupconsisting of sequences of odd SEQ ID NOs:1-23 and sequences of cloneinserts of the deposited clone pool, using information from thesequences disclosed herein or the clone pool deposited with the ATCC.For example, allelic variants, orthologs and/or species homologues maybe isolated and identified by making suitable probes or primers from thesequences provided herein and screening a suitable nucleic acid sourcefor allelic variants and/or the desired homologue using any techniqueknown to those skilled in the art including those described into thesection entitled “To find similar sequences”.

In a specific embodiment, the polynucleotides of the invention are atleast 15, 30, 50, 100, 125, 500, or 1000 continuous nucleotides. Inanother embodiment, the polynucleotides are less than or equal to 300kb, 200 kb, 100 kb, 50 kb, 10 kb, 7.5 kb, 5kb, 2.5 kb, 2 kb, 1.5 kb, or1kb in length. In a further embodiment, polynucleotides of the inventioncomprise a portion of the coding sequences, as disclosed herein, but donot comprise all or a portion of any intron. In another embodiment, thepolynucleotides comprising coding sequences do not contain codingsequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene ofinterest in the genome). In other embodiments, the polynucleotides ofthe invention do not contain the coding sequence of more than 1000, 500,250, 100, 75, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 naturally occurringgenomic flanking gene(s).

Deposited Clone Pool of the Invention

Expression of GENSET genes has been shown to lead to the production ofat least one mRNA species per GENSET gene, which cDNA sequence is setforth in the appended Sequence Listing as odd SEQ ID NOs:1-23. The cDNAscorresponding to these GENSET mRNA species were cloned either in thevector pBluescriptII SK⁻ (Stratagene) or in a vector called pPT. Cellscontaining the cloned cDNAs of the present invention are maintained inpermanent deposit by the inventors at Genset, S. A., 24 Rue Royale,75008 Paris, France. Table I provides Genset's internal designationnumber assigned to each SEQ ID NO., and indicates whether the sequenceis a nucleic acid sequence (DNA) or a protein (PRT) sequence. Each cDNAcan be removed from the Bluescript vector in which it was inserted byperforming a NotI Pst I double digestion, or from the pPT vector byperforming a MunI HindIII double digestion, to produce the appropriatefragment for each clone, provided the cDNA sequence does not contain anyof the corresponding restriction sites within its sequence.Alternatively, other restriction enzymes of the multicloning site of thevector may be used to recover the desired insert as indicated by themanufacturer.

Pools of cells containing GENSET genes as described in the SequenceListing, from which the cells containing a particular polynucleotide isobtainable, were or will be also deposited with the American TissueCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110-2209, United States. Each cDNA clone has been transfected intoseparate bacterial cells (E-coli) for these composite deposits.

Bacterial cells containing a particular clone can be obtained from thecomposite deposit using standard methods, e.g., by plating a culture ofthe composite deposit, transferring the lysed colonies to a filter, andhybridizing the filter with a labelled probe such as an oligonucleotideprobe specific for the clone of interest. Alternatively, individual cDNAinserts can be recovered from the pool of bacteria using PCR withprimers designed at both ends of the cDNA insertion, including primersdesigned in the multicloning site of the vector.

cDNA Sequences of the Invention

Structural parameters of each of the cDNAs of the present invention arepresented in the appended Sequence Listing. Accordingly, the codingsequence (CDS) or open reading frame (ORF) of each cDNA of the inventionrefers to the nucleotide sequence beginning with the first nucleotide ofthe start codon and ending with the nucleotide immediately 5′ to thefirst nucleotide of the stop codon. Similarly, the 5′ untranslatedregion (or 5′UTR) of each cDNA of the invention refers to the nucleotidesequence starting at nucleotide 1 and ending at the nucleotideimmediately 5′ to the first nucleotide of the start codon. The 3′untranslated region (or 3′UTR) of each cDNA of the invention refers tothe nucleotide sequence starting at the first nucleotide of the stopcodon and ending at the last nucleotide of the cDNA.

Coding Sequences

Another object of the invention is an isolated, purified or recombinantpolynucleotide comprising the coding sequence of a sequence selectedfrom the group consisting of the polynucleotide sequences of theappended Sequence Listing, those of human cDNA clone inserts of thedeposited clone pool and variants thereof.

It will be appreciated that should the extent of the coding sequencediffer from that indicated in the appended sequence listing as a resultof a sequencing error, reverse transcription or amplification error,mRNA splicing, post-translational modification of the encoded protein,enzymatic cleavage of the encoded protein, or other biological factors,one skilled in the art would be readily able to identify the extent ofthe coding sequences in the polynucleotide sequences of the SequenceListing, those of the human cDNA inserts of the deposited clone pool,and allelic variants thereof. Accordingly, the scope of any claimsherein relating to nucleic acids containing the coding sequence of oneof the polynucleotide sequences of the Sequence Listing and those of thecDNA inserts of the deposited clone pool is not to be construed asexcluding any readily identifiable variations from or equivalents to thecoding sequences described in the appended sequence listing. Equivalentsinclude any alterations in a nucleotide coding sequence that does notresult in an amino acid change, or that results in a conservative aminoacid substitution, as defined below, in the polypeptide encoded by thenucleotide sequence. Similarly, should the extent of the polypeptidesdiffer from those indicated in the appended Sequence Listing as a resultof any of the preceding factors, the scope of claims relating topolypeptides comprising the amino acid sequence of the polypeptidesequences of the appended Sequence Listing is not to be construed asexcluding any readily identifiable variations from or equivalents to thesequences described in the appended sequence listing.

The above disclosed polynucleotides that contain the coding sequence ofthe GENSET genes may be expressed in a desired host cell or a desiredhost organism, when this polynucleotide is placed under the control ofsuitable expression signals. The expression signals may be either theexpression signals contained in the regulatory regions in the GENSETgenes of the invention or, in contrast, the signals may be exogenousregulatory nucleic sequences. Such a polynucleotide, when placed underthe suitable expression signals, may also be inserted in a vector forits expression and/or amplification.

Further included in the present invention are polynucleotides encodingthe polypeptides of the present invention that are fused in frame to thecoding sequences for additional heterologous amino acid sequences. Alsoincluded in the present invention are nucleic acids encodingpolypeptides of the present invention together with additional,non-coding sequences, including, but not limited to, non-coding 5′ and3′ sequences, vector sequence, sequences used for purification, probing,or priming. For example, heterologous sequences include transcribed,untranslated sequences that may play a role in transcription and mRNAprocessing, such as ribosome binding and stability of mRNA. Theheterologous sequences may alternatively comprise additional codingsequences that provide additional functionalities, e.g. a hexahistidineor HA tag).

Regulatory Sequences of the Invention

As mentioned, the genomic sequence of GENSET genes contain regulatorysequences in the non-coding 5′-flanking region and possibly in thenon-coding 3′-flanking region that border the GENSET polypeptide codingregions containing the exons of these genes.

Polynucleotides derived from GENSET polynucleotide 5′ and 3′ regulatoryregions are useful in order to detect the presence of at least a copy ofa genomic nucleotide sequence of the GENSET gene or a fragment thereofin a test sample.

Preferred Regulatory Sequences

Polynucleotides carrying the regulatory elements located at the 5′ endand at the 3′ end of GENSET polypeptide coding regions may beadvantageously used to control the processing, localization, stability,maturation and transcriptional and translational activity of aheterologous polynucleotide of interest, e.g; the regulatorypolynucleotides may be part of a recombinant expression vector that maybe used to express a coding sequence in a desired host cell or hostorganism (for a review on UTRs see Decker and Parker, (1995) Curr. Opin.Cell. Biol. 7(3):368-92, Derrigo et al., (2000) Int. J. Mol. Med.5(2):111-23). In particular, 3′UTRs may be used in order to control thestability of heterologous mRNAs in recombinant vectors using any methodsknown to those skilled in the art including Makrides (1999) Protein ExprPurif 1999 November; 17(2):183-202), U.S. Pat. Nos. 5,925,564; 5,807,707and 5,756,264.

The present invention also concerns a purified or isolated nucleic acidcomprising a polynucleotide which is selected from the group consistingof the 5′ and 3′ GENSET polynucleotide regulatory regions, sequencescomplementary thereto, regulatory active fragments and variants thereof,and those that hybridize under stringent hybridization conditionstherewith. Further included are nucleic acids comprising a nucleotidesequence having at least 95% identity with any of the herein-describedGENSET 5′ or 3′ regulatory sequences, as well as 5′- or 3′-UTRs of thepolynucleotide sequences of the appended Sequence Listing, of human cDNAclone inserts of the deposited clone pool, sequences complementarythereto, regulatory active fragments and allelic variants thereof.Fragments of 5′ and 3′ regulatory regions may have a lengthcorresponding to any one integer between 20 and 20,000 nucleotides inlength.

For the purpose of the invention, a nucleic acid or polynucleotide is“functional” as a “regulatory region” for expressing a recombinantpolypeptide or a recombinant polynucleotide if said regulatorypolynucleotide contains nucleotide sequences which containtranscriptional and translational regulatory information, and suchsequences are “operably linked” to nucleotide sequences which encode thedesired polypeptide or the desired polynucleotide.

The invention also comprises a nucleic acid molecule encoding a desiredpolypeptide or a nucleic acid molecule of interest, wherein said nucleicacid molecule is operably linked to any of the herein-describedregulatory sequences. The desired polypeptide may be of various natureor origin, encompassing proteins of prokaryotic viral or eukaryoticorigin. Also encompassed are eukaryotic proteins such as intracellularproteins, such as “house keeping” proteins, membrane-bound proteins,such as mitochondrial membrane-bound proteins and cell surfacereceptors, and secreted proteins such as endogenous mediators such ascytokines. The desired polypeptide may be a heterologous polypeptide ora GENSET polypeptide.

Polynucleotide Variants

The invention also relates to variants of the polynucleotides describedherein and fragments thereof. “Variants” of polynucleotides, as the termis used herein, are polynucleotides that differ from a referencepolynucleotide. Generally, differences are limited so that thenucleotide sequences of the reference and the variant are closelysimilar overall and, in many regions, identical. The present inventionencompasses both allelic variants and degenerate variants.

Allelic Variant

A variant of a polynucleotide may be a naturally occurring variant suchas a naturally occurring allelic variant, or it may be a variant that isnot known to occur naturally. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism (see Lewin, (1989), PNAS 86:9832-8935).Diploid organisms may be homozygous or heterozygous for an allelic form.Non-naturally occurring variants of the polynucleotide may be made byart-known mutagenesis techniques, including those applied topolynucleotides, cells or organisms.

Degenerate Variant

In addition to the isolated polynucleotides of the present invention,and fragments thereof, the invention further includes polynucleotideswhich comprise a sequence substantially different from those describedabove but which, due to the degeneracy of the genetic code, still encodea GENSET polypeptide of the present invention. These polynucleotidevariants are referred to as “degenerate variants” throughout the instantapplication. That is, all possible polynucleotide sequences that encodethe GENSET polypeptides of the present invention are contemplated. Thisincludes the genetic code and species-specific codon preferences knownin the art.

Nucleotide changes present in a variant polynucleotide may be silent,which means that they do not alter the amino acids encoded by thepolynucleotide. However, nucleotide changes may also result in aminoacid substitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence. The substitutions,deletions or additions may involve one or more nucleotides. The variantsmay be altered in coding or non-coding regions or both. Alterations inthe coding regions may produce conservative or non-conservative aminoacid substitutions, deletions or additions. In the context of thepresent invention, preferred embodiments are those in which thepolynucleotide variants encode polypeptides which retain substantiallythe same biological properties or activities as the GENSET protein. Morepreferred polynucleotide variants are those containing conservativesubstitutions.

Similar Polynucleotides

Other embodiments of the present invention provide a purified, isolatedor recombinant polynucleotide which is at least 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identical to a polynucleotide of the present invention.Although similar polynucleotides encoding polypeptides with GENSETbiological activity are preferred, the presence of GENSET biologicalactivity in an encoded protein is not necessary because even where aparticular nucleic acid molecule does not encode a polypeptide havingactivity, one of skill in the art would still know how to use thenucleic acid molecule, for instance, as a hybridization probe or primer.Uses of the nucleic acid molecules of the present invention that do notencode a polypeptide having GENSET activity include, inter alia,isolating a GENSET gene or allelic variants thereof from a DNA library,and detecting GENSET mRNA expression in biological samples suspected ofcontaining GENSET mRNA or DNA, e.g., by Northern Blot or PCR analysis.

Hybridizing Polynucleotides

In another aspect, the invention provides an isolated or purifiednucleic acid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to any polynucleotide of the present.Such hybridizing polynucleotides may be of at least any one integerbetween 10 and 10,000 nucleotides in length.

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA shown in the sequencelisting), or to a 5′ complementary stretch of T (or U) residues, wouldnot be included in the definition of “polynucleotide,” since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly(A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer).

Complementary Polynucleotides

The invention further provides isolated nucleic acid molecules having anucleotide sequence fully complementary to any polynucleotide of theinvention.

Polynucleotide Fragments

The present invention is further directed to portions or fragments ofthe polynucleotides of the present invention. Uses for thepolynucleotide fragments of the present invention include probes,primers, molecular weight markers and for expressing the polypeptidefragments of the present invention. Fragments include portions ofpolynucleotides selected from the group consisting of a) polynucleotidesequences of the Sequence Listing, b) genomic GENSET sequences, c)polynucleotides encoding a polypeptide of the present invention, d)sequences of human cDNA clone inserts of the deposited clone pool, ande) polynucleotides encoding the polypeptides encoded by the human cDNAclone inserts of the deposited clone pool. Particularly included in thepresent invention is a purified or isolated polynucleotide comprising atleast 8, 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200,300, 400, 500, 800, 1000, 1500, or 2000 consecutive nucleotides of apolynucleotide of the present invention. Further, polynucleotides areprovided comprising at least X nucleotides, wherein “X” is defined asany integer between 8 and the integer representing the 3′ mostnucleotide position as set forth in the sequence listing or elsewhereherein.

Further included as preferred polynucleotides of the present inventionare polynucleotide fragments that are specified in terms of their 5′ and3′ position. Where the 5′ and 3′ positions are represented by theposition numbers set forth in the appended sequence listing wherein the5′ most nucleotide is 1 and the 3′ most nucleotide is the lastnucleotide for a particular SEQ ID No., all polynucleotide fragmentscorresponding to every combination of a 5′ and 3′ nucleotide positionthat a polynucleotide fragment of the present invention, at least 8contiguous nucleotides in length, could occupy on a polynucleotide ofthe invention are specifically considered. These species ofpolynucleotide fragments may alternatively be described by the formula“a to b”; where “a” equals the 5′ most nucleotide position and “b”equals the 3′ most nucleotide position of the polynucleotide; andfurther where “a” equals an integer between 1 and the number ofnucleotides of the polynucleotide sequence of the present inventionminus 8, and where “b” equals an integer between 9 and the number ofnucleotides of the polynucleotide sequence of the present invention; andwhere “a” is an integer smaller then “b” by at least 8. All of thepolynucleotide fragments described in either of these ways can beimmediately envisaged and are therefore not individually listed solelyfor the purpose of not unnecessarily lengthening the specification. Anyof these polynucleotide fragments may also be specifically excluded fromthe present invention. Any number of fragments specified by 5′ and 3′positions or by size in nucleotides, as described above, may beexcluded. Preferred excluded fragments include those having substantialhomology to repeated sequences including Alu, L1, THE and MER repeats,SSTR sequences or satellite, micro-satellite, and telomeric repeats.

Other preferred fragments of the invention are polynucleotidescomprising polynucleotide sequences encoding domains of polypeptides.Such fragments may be used to obtain other polynucleotides encodingpolypeptides having similar domains using hybridization or RT-PCRtechniques. Alternatively, these fragments may be used to express apolypeptide domain which may have a specific biological property.

Another object of the invention is an isolated, purified or recombinantpolynucleotide encoding a polypeptide consisting of, consistingessentially of, or comprising a contiguous span of at least (any integerbetween 5 and 1,000 consecutive amino acids in length more preferably atleast) 5, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150 or200 consecutive amino acids.

The present invention further encompasses any combination of thepolynucleotide fragments listed in this section.

Oligonucleotide Primers and Probes

The present invention also encompasses fragments of GENSETpolynucleotides for use as primers and probes. Polynucleotides derivedfrom the GENSET genomic and cDNA sequences are useful in order to detectthe presence of at least a copy of a GENSET polynucleotide or fragment,complement, or variant thereof in a test sample.

Structural Definition

Any polynucleotide of the invention may be used as a primer or probe.Particularly preferred probes and primers of the invention includeisolated, purified, or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides of a polynucleotide ofthe present invention.

For amplification purposes, pairs of primers with approximately the sameTm are preferable. Primers may be designed using methods known in theart. Amplification techniques that can be used in the context of thepresent invention include, but are not limited to, the ligase chainreaction (LCR) described in EP-A-320 308, WO 9320227 and EP-A-439 182,the polymerase chain reaction (PCR, RT-PCR) and techniques such as thenucleic acid sequence based amplification (NASBA) described in Guatelliet al., (1990) PNAS 35:273-286 and in Compton (1991) Nature350(6313):91-92, Q-beta amplification as described in European PatentApplication No 4544610, strand displacement amplification as describedin Walker, et al. (1996), Clin. Chem. 42:9-13 and EP A 684 315 and,target mediated amplification as described in WO 9322461.

The probes of the present invention are useful for a number of purposes,including for Southern hybridization to genomic DNA, to detect PCRamplification products, to detect mismatches in a GENSET gene or mRNA,and in in situ hybridization. Any of the polynucleotides, primers andprobes of the present invention can be conveniently immobilized on anytype of solid support, such as latex particles, microparticles, magneticbeads, non-magnetic beads (including polystyrene beads), membranes(including nitrocellulose strips), plastic tubes, walls of microtiterwells, glass or silicon chips, sheep (or other suitable animal's) redblood cells and duracytes. Suitable methods for immobilizing nucleicacids on solid phases include ionic, hydrophobic, covalent interactionsand the like. A solid support, as used herein, refers to any materialwhich is insoluble, or can be made insoluble by a subsequent reaction.The solid support can be chosen for its intrinsic ability to attract andimmobilize the capture reagent, or alternatively may retain anadditional receptor which has the ability to attract and immobilize thecapture reagent. The polynucleotides of the invention can be attached toor immobilized on a solid support individually or in groups of at least2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the inventionto a single solid support. In addition, polynucleotides other than thoseof the invention may be attached to the same solid support as one ormore polynucleotides of the invention.

Oligonucleotide Array

A substrate comprising a plurality of oligonucleotide primers or probesof the invention may be used either for detecting or amplifying targetedsequences in GENSET genes, may be used for detecting mutations in thecoding or in the non-coding sequences of GENSET genes, and may also beused to determine GENSET gene expression in different contexts such asin different tissues, at different stages of a process (embryodevelopment, disease treatment), and in patients versus healthyindividuals as described elsewhere in the application.

As used herein, the term “array” means a one dimensional, twodimensional, or multidimensional arrangement of nucleic acids ofsufficient length to permit specific detection of gene expression. Forexample, the array may contain a plurality of nucleic acids derived fromgenes whose expression levels are to be assessed. The array may includeany of the herein-described GENSET genomic DNA, a GENSET cDNA, sequencescomplementary thereto or fragments thereof. Preferably, the fragmentsare at least 12, 15, 18, 20, 25, 30, 35, 40, 50, or 100 nucleotides inlength.

Any polynucleotide provided herein may be attached in overlapping areasor at random locations on the solid support. Alternatively thepolynucleotides of the invention may be attached in an ordered arraywherein each polynucleotide is attached to a distinct region of thesolid support which does not overlap with the attachment site of anyother polynucleotide. Preferably, such an ordered array ofpolynucleotides is designed to be “addressable” where the distinctlocations are recorded and can be accessed as part of an assayprocedure. Addressable polynucleotide arrays typically comprise aplurality of different oligonucleotide probes that are coupled to asurface of a substrate in different known locations. The knowledge ofthe precise location of each polynucleotides location makes these“addressable” arrays particularly useful in hybridization assays. Anyaddressable array technology known in the art can be employed with thepolynucleotides of the invention, including Geneclips™ (see, e.g., U.S.Pat. No. 5,143,854; PCT publications WO 90/15070 and 92/10092, Fodor etal., (1991) Science 251:767-777). The immobilization of arrays ofoligonucleotides on solid supports has been rendered possible by thedevelopment of a technology generally identified as “Very Large ScaleImmobilized Polymer Synthesis” (VLSIPS™) in which, typically, probes areimmobilized in a high density array on a solid surface of a chip (see,e.g., U.S. Pat. Nos. 5,143,854 and 5,412,087, and in PCT Publications WO90/15070, WO 92/10092 and WO 95/11995). Further presentation strategiesknown in the art may be used, such as those disclosed in WO 94/12305, WO94/11530, WO 97/29212 and WO 97/31256.

Consequently, the invention concerns an array of nucleic acid moleculescomprising at least one, two, or five polynucleotides of the invention,particularly probes or primers as described herein.

Methods of Making the Polynucleotides of the Invention

The present invention also comprises methods of making thepolynucleotides of the invention. Polynucleotides of the invention maybe synthesized either enzymatically using techniques well known to thoseskilled in the art including amplification or hybridization-basedmethods as described herein, or chemically.

A variety of chemical methods of synthesizing nucleic acids are known tothose skilled in the art. In many of these methods, synthesis isconducted on a solid support. Alternatively, polynucleotides may beprepared as described in U.S. Pat. No. 5,049. In some embodiments,several polynucleotides prepared as described above are ligated togetherto generate longer polynucleotides having a desired sequence.

Polypeptides of the Invention

The term “GENSET polypeptides” is used herein to embrace all of theproteins and polypeptides of the present invention. The presentinvention encompasses GENSET polypeptides, including recombinant,isolated or purified GENSET polypeptides consisting of: (a) the fulllength polypeptides of even SEQ ID NOs:2-24; (b) the full lengthpolypeptides encoded by the clone inserts of the deposited clone pool;(c) the epitope-bearing fragments of the polypeptides of even SEQ IDNOs:2-24; (d) the epitope-bearing fragments of the polypeptides encodedby the clone inserts contained in the deposited clone pool; (e) thedomains of the polypeptides of even SEQ ID NOs:2-24; (f) the domains ofthe polypeptides encoded by the clone inserts contained in the depositedclone pool; (g) the signal peptides of the polypeptides of even SEQ IDNOs:2-24 or encoded by the human cDNAs of the deposited clone pool; (h)the mature polypeptides of even SEQ ID NOs:2-24 or encoded by the humancDNAs of the deposited clone pool; and (i) the allelic variantpolypeptides of any of the polypeptides of (a)-(f). Other objects of theinvention are polypeptides encoded by the polynucleotides of theinvention as well as fusion polypeptides comprising such polypeptides.

Polypeptide Variants

The present invention further provides for GENSET polypeptides encodedby allelic and splice variants, orthologs, and/or species homologues.Procedures known in the art can be used to obtain, allelic variants,splice variants, orthologs, and/or species homologues of polynucleotidesencoding polypeptides of the Sequence Listing and polypeptides encodedby the clone inserts of the deposited clone pool, using information fromthe sequences disclosed herein or the clones deposited with the ATCC.

The polypeptides of the present invention also include polypeptideshaving an amino acid sequence at least 50% identical, more preferably atleast 60% identical, and still more preferably 70%, 80%, 90%, 95%, 96%,97%, 98% or 99% identical to a polypeptide of the present invention. Bya polypeptide having an amino acid sequence at least, for example, 95%“identical” to a query amino acid sequence of the present invention, itis intended that the amino acid sequence of the subject polypeptide isidentical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% (5 of 100) of the amino acidresidues in the subject sequence may be inserted, deleted, (indels) orsubstituted with another amino acid.

Further polypeptides of the present invention include polypeptides whichhave at least 90% similarity, more preferably at least 95% similarity,and still more preferably at least 96%, 97%, 98% or 99% similarity tothose described above. “Similarity” is calculated exactly as describedabove for “identity”, except that for the purposes of the calculation amatching amino acid can be either identical or an amino acidrepresenting an “equivalent” change, as defined below.

These alterations of the reference sequence may occur at the amino orcarboxy terminal positions of the reference amino acid sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. The variantpolypeptides described herein are included in the present inventionregardless of whether they have their normal biological activity, as oneof skill in the art would recognize that variant polypeptides lackingbiological activity would still be useful, for instance, as a vaccine,to generate antibodies, as epitope tags, in epitope mapping, asmolecular weight markers on SDS-PAGE gels or on molecular sieve gelfiltration columns.

Preparation of the Polypeptides of the Invention

The polypeptides of the present invention can be prepared in anysuitable manner known in the art. Such polypeptides include isolatednaturally occurring polypeptides, recombinantly produced polypeptides,synthetically produced polypeptides, or polypeptides produced by acombination of these methods. The polypeptides of the present inventionare preferably provided in an isolated form, and may be partially orpreferably substantially purified.

Isolation

From Natural Sources

The GENSET proteins of the invention may be isolated from naturalsources, including bodily fluids, tissues and cells, whether directlyisolated or cultured cells, of humans or non-human animals. Methods forextracting and purifying natural proteins are known in the art, andinclude the use of detergents or chaotropic agents to disrupt particlesfollowed by differential extraction and separation of the polypeptidesby ion exchange chromatography, affinity chromatography, sedimentationaccording to density, and gel electrophoresis. See, e.g., “Methods inEnzymology, Academic Press, 1993” for a variety of methods for purifyingproteins. Polypeptides of the invention also can be purified fromnatural sources using antibodies directed against the polypeptides ofthe invention, using standard methods.

From Recombinant Sources

Preferably, the GENSET polypeptides of the invention are recombinantlyproduced using routine expression methods known in the art. Thepolynucleotide encoding the desired polypeptide is operably linked to apromoter into an expression vector suitable for any convenient host.Both eukaryotic and prokaryotic host systems are used in formingrecombinant polypeptides. The polypeptide is then isolated from lysedcells or from the culture medium and purified to the extent needed forits intended use. Any polynucleotide of the present invention may beused to express GENSET polypeptides.

Consequently, a further embodiment of the present invention is a methodof making a polypeptide of the present invention, said method comprisingthe steps of providing a GENSET polynucleotide (e.g. a polynucleotideencoding a GENSET polypeptide), inserting the polynucleotide in anexpression vector such that the cDNA is operably linked to a promoter;and introducing the expression vector into a host cell whereby said hostcell produces said polypeptide. In one aspect of this embodiment, themethod further comprises the step of isolating the polypeptide. Anysuitable expression vector and system (e.g. cell-based system such as3T3 cells) may be used, according to methods well known in the art.

In one embodiment, the entire coding sequence of a GENSET cDNA and the3′UTR through the polyA signal of the cDNA is operably linked to apromoter in the expression vector.

In another embodiment, an additional nucleotide sequence is includedwhich codes for secretory or leader sequences, pro-sequences, sequenceswhich aid in purification, such as multiple histidine residues, or anadditional sequence for stability during recombinant production.

Alternatively, the GENSET polypeptide to be expressed may also be aproduct of transgenic animals, i.e., as a component of the milk oftransgenic cows, goats, pigs or sheep which are characterized by somaticor germ cells containing a nucleotide sequence encoding the protein ofinterest.

Any standard method may be used to recover a GENSET polypeptideexpressed using these methods, including differential extraction,ammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, antibody-based methods, affinitychromatography, hydroxylapatite chromatography, HPLC,immunochromatography, and lectin chromatography.

Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benor-glycosylated, and may also include an initial modified methionineresidue, in some cases as a result of host-mediated processes. Inaddition, polypeptides of the present invention may or may not containthe amino terminal methionine.

From Chemical Synthesis

In addition, polypeptides of the invention, especially short proteinfragments, can be chemically synthesized using techniques known in theart (See, e.g., Creighton (1983), Proteins: Structures and MolecularPrinciples, W.H. Freeman & Co. 2nd Ed., T. E., New York; and Hunkapilleret al., (1984) Nature. 310(5973):105-11). For example, a polypeptidecorresponding to a fragment of a polypeptide sequence of the inventioncan be synthesized by use of a peptide synthesizer. Alternatively, themethods described in U.S. Pat. No. 5,049,656, may be used.

Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thepolypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

Modifications

The invention encompasses polypeptides which are differentially modifiedduring or after translation, e.g., by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. Any of numerous chemical modifications may becarried out by known techniques, including, but not limited to, specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH4; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends, attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of prokaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see, e.g., U.S. Pat.No. 4,179,337). The chemical moieties for derivatization may be selectedfrom water soluble polymers (branched or unbranched) such aspolyethylene glycol (preferably between 1 and 100 kD), ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties. The chemical moiety may be attached using any method,e.g. via a free amino, carboxyl, or sulfhydryl group (see, e.g., EP 0401 384, or Malik et al., (1992), Exp. Hematol. 20:1028-1035).

Multimerization

The polypeptides of the invention may be in monomers or multimers (i.e.,dimers, trimers, tetramers and higher multimers). Accordingly, thepresent invention relates to monomers and multimers of the polypeptidesof the invention, their preparation, and compositions containing them.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term “homomer” refers to a multimer containing onlypolypeptides with the same amino acid sequence (although a small amountof variation is allowed), and “heteromer” refers to a multimercontaining one or more heterologous polypeptides.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked. Thus, in one embodiment, multimers of the invention, such as,for example, homodimers or homotrimers, are formed when polypeptides ofthe invention contact one another in solution. In another embodiment,heteromultimers of the invention, such as, for example, heterotrimers orheterotetramers, are formed when polypeptides of the invention contactantibodies to the polypeptides of the invention (including antibodies tothe heterologous polypeptide sequence in a fusion protein of theinvention) in solution. In other embodiments, multimers of the inventionare formed by cross-linking between cysteine residues located within thepolypeptide sequences. Any of these associations may involve one or moreamino acid residues contained in an amino acid provided in the sequencelisting or in a heterologous polypeptide sequence of a fusion protein,e.g., in an Fc fusion protein, osteoprotegerin fusion protein, peptidelinker fusion protein, Flag® fusion protein, or leucine zipper fusionprotein of the invention (see, e.g., U.S. Pat. No. 5,478,925, WO98/49305, or U.S. Pat. No. 5,073,627, Landschulz et al., (1988),Science. 240:1759, WO 94/10308, Hoppe et al., (1994), FEBS Letters.344:191 and in U.S. patent application Ser. No. 08/446,922). Othermethods of making multimers include the addition of cysteine or biotinto the C-terminus or N-terminus of the polypeptide using techniquesknown in the art, or by generating liposomes containing the polypeptidecomponents desired to be contained in the multimer of the invention(see, e.g., U.S. Pat. No. 5,478,925 for numerous methods ofmultimerization).

Multimers of the invention may be generated using chemical or geneticengineering techniques known in the art.

Mutated Polypeptides

To improve or alter the characteristics of GENSET polypeptides of thepresent invention, recombinant DNA technology can be used to createnovel mutant proteins or muteins including single or multiple amino acidsubstitutions, deletions, additions, or fusion proteins. Such modifiedpolypeptides can show, e.g., increased/decreased biological activity orincreased/decreased stability. In addition, they may be purified inhigher yields and show better solubility than the corresponding naturalpolypeptide, at least under certain purification and storage conditions.

N- and C-Terminal Deletions

It is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. (See, e.g., Ron et al., (1993), Biol. Chem., 268 2984-2988;Dobeli, et al. 1988.) Accordingly, the present invention providespolypeptides having one or more residues deleted from the amino and/orcarboxy terminus.

Other Mutations

The invention includes numerous variations of the GENSET polypeptideswhich show substantial GENSET polypeptide activity. Such mutants includedeletions, insertions, inversions, repeats, and substitutions selectedaccording to general rules known in the art so as to have little effecton activity.

There are two main approaches for studying the tolerance of an aminoacid sequence to change (see, Bowie et al., (1994), Science.247:1306-1310). The first method relies on the process of evolution, inwhich mutations are either accepted or rejected by natural selection.The second approach uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene and selections or screensto identify sequences that maintain functionality. Examples of thisinclude site-directed mutagenesis or alanine-scanning mutagenesis (see,e.g., Cunningham et al. (1989), Science 244:1081-1085). These studieshave revealed that proteins are surprisingly tolerant of amino acidsubstitutions.

Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu and Phe;interchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu, substitution between the amide residues Asn andGln, exchange of the basic residues Lys and Arg and replacements amongthe aromatic residues Phe, Tyr. Thus, the polypeptide of the presentinvention may be, for example, one in which one or more of the aminoacid residues are substituted with a conserved or non-conserved aminoacid residue (preferably a conserved amino acid residue). Suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, and may include a substituent group.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein. The following groups of aminoacids represent equivalent changes: (1) Ala, Pro, Gly, Glu, Asp, Gln,Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, Ile, Leu, Met, Ala, Phe;(4) Lys, Arg, His; (5) Phe, Tyr, Trp, His.

Furthermore, GENSET polypeptides of the present invention may includeone or more amino acid substitutions that mimic modified amino acids. Anexample of this type of substitution includes replacing amino acids thatare capable of being phosphorylated (e.g., serine, threonine, ortyrosine) with a negatively charged amino acid that resembles thenegative charge of the phosphorylated amino acid (e.g., aspartic acid orglutamic acid). Also included is substitution of amino acids that arecapable of being modified by hydrophobic groups (e.g., arginine) withamino acids carrying bulky hydrophobic side chains, such as tryptophanor phenylalanine. Therefore, a specific embodiment of the inventionincludes GENSET polypeptides that include one or more amino acidsubstitutions that mimic modified amino acids at positions where aminoacids that are capable of being modified are normally positioned.Furthermore, any GENSET polypeptide amino acid capable of being modifiedmay be excluded from substitution with a modification-mimicking aminoacid.

A specific embodiment of a modified GENSET peptide molecule of interestaccording to the present invention, includes, but is not limited to, apeptide molecule which is resistant to proteolysis, is a peptide inwhich the —CONH— peptide bond is modified and replaced by a (CH2NH)reduced bond, a (NHCO) retro inverso bond, a (CH2—O) methylene-oxy bond,a (CH2—S) thiomethylene bond, a (CH2CH2) carba bond, a (CO—CH2)cetomethylene bond, a (CHOH—CH2) hydroxyethylene bond), a (N—N) bound, aE-alcene bond or also a —CH═CH— bond.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic (see, e.g., Pinckard et al., (1967), Clin. Exp. Immunol2:331-340; Robbins et al., (1987), Diabetes. 36:838-845; and Cleland etal., (1993) Crit. Rev. Ther. Drug Carr. Syst. 10:307-377).

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of a GENSET polypeptide having anamino acid sequence which contains at least any one integer from 1 to 50of conservative amino acid substitutions. Any conservative substitutionor combination of substitutions may also be excluded.

Polypeptide Fragments

Structural Definition

The present invention is further directed to fragments of thepolypeptides of the present invention. More specifically, the presentinvention embodies purified, isolated, and recombinant polypeptidescomprising at least any one integer between 6 and 1000 (or the length ofthe polypeptides amino acid residues minus 1 if the length is less than1000) of consecutive amino acid residues. Preferably, the fragments areat least 6, preferably at least 8 to 10, more preferably 12, 15, 20, 25,30, 35, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300consecutive amino acids of a polypeptide of the present invention.

In addition to the above polypeptide fragments, further preferredsub-genuses of polypeptides comprise at least X amino acids, wherein “X”is defined as any integer between 6 and the integer representing theC-terminal amino acid of the polypeptide of the present inventionincluding the polypeptide sequences of the sequence listing below.Further included are species of polypeptide fragments at least 6 aminoacids in length, as described above, that are further specified in termsof their N-terminal and C-terminal positions. However, included in thepresent invention as individual species are all polypeptide fragments,at least 6 amino acids in length, as described above, and may beparticularly specified by a N-terminal and C-terminal position. That is,every combination of a N-terminal and C-terminal position that afragment at least 6 contiguous amino acid residues in length couldoccupy, on any given amino acid sequence of the sequence listing or ofthe present invention is included in the present invention

Further preferred polypeptide fragments comprising amino acids of thesequences of the EVEN numbered SEQ ID NOs. of the Sequence listing, andpolynucleotides encoding the same, are selected from the groupconsisting of amino acids starting at position one and continuing to anyposition selected from the group consisting of 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339,340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437,438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493,494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507,508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521,522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535,536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549,550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563,564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577,578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591,592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605,606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619,620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633,634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647,648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661,662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675,676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689,690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703,704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717,718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731,732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745,746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759,760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773,774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, and 786,wherein the numbering of amino acids comprising any one fragment isconsistent with the polypeptide sequence of any one EVEN numbered SEQ IDof the Sequence listing.

Further preferred polypeptide fragments comprising amino acids of thesequences of the EVEN numbered SEQ ID NOs. of the Sequence listing, andpolynucleotides encoding the same, are selected from the groupconsisting of amino acids ending at the terminal amino acid of theprotein (e.g. position 787) and beginning at any position selected fromthe group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285,286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313,314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327,328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341,342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355,356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383,384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397,398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411,412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425,426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439,440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467,468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481,482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495,496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509,510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523,524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537,538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551,552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565,566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579,580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593,594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607,608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621,622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635,636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649,650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663,664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677,678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691,692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705,706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719,720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733,734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747,748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761,762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775,776, 777, 778, 779, 780, 781, and 782, wherein the numbering of aminoacids comprising any one fragment is consistent with the polypeptidesequence of any one EVEN numbered SEQ ID of the Sequence listing.

Further preferred polypeptide fragments of the EVEN numbered SEQ ID NOs.of the Sequence listing, and polynucleotides encoding the same, areselected from the group consisting of fragments comprising any 50 or 100consecutive amino acids starting from an amino acid position selectedfrom the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256,257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298,299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326,327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340,341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354,355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368,369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382,383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396,397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424,425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438,439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452,453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466,467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480,481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494,495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508,509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522,523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536,537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550,551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564,565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578,579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592,593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606,607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620,621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634,635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648,649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662,663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676,677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690,691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704,705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718,719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732,733, 734, 735, 736, 737, and 738, wherein the numbering of amino acidscomprising any one fragment is consistent with the polypeptide sequenceof any one EVEN numbered SEQ ID of the Sequence listing.

These specific embodiments, and other polypeptide and polynucleotidefragment embodiments described herein may be modified as being “atleast_”, “equal to”, “equal to or less than”, “less than”, “at least_butnot greater than_” or “from_to_”. a specified size or specifiedN-terminal and/or C-terminal positions. It is noted that all ranges usedto describe any embodiment of the present invention are inclusive unlessspecifically set forth otherwise.

The above species of polypeptide fragments of the present invention mayalternatively be described by the formula “a to b”; where “a” equals theN-terminal most amino acid position and “b” equals the C-terminal mostamino acid position of the polynucleotide; and further where “a” equalsan integer between 1 and the number of amino acids of the polypeptidesequence of the present invention minus 6, and where “b” equals aninteger between 7 and the number of amino acids of the polypeptidesequence of the present invention; and where “a” is an integer smallerthen “b” by at least 6.

The above polypeptide fragments of the present invention can beimmediately envisaged using the above description and are therefore notindividually listed solely for the purpose of not unnecessarilylengthening the specification. Moreover, the above fragments need nothave a GENSET biological activity, although polypeptides having theseactivities are preferred embodiments of the invention, since eveninactive fragments are useful, for example, in immunoassays, in epitopemapping, epitope tagging, as vaccines, as molecular weight markers, andto generate antibodies to a particular portion of the polypeptide.

The present invention also provides for the exclusion in any polypeptideof any one or more of the above-described fragments, e.g., one or moreindividual fragments specified by N-terminal and C-terminal positions orof any fragments specified by size in amino acid residues as describedabove.

Functional Definition

Domains

Preferred polynucleotide fragments of the invention comprise domains ofpolypeptides of the invention. Such domains may eventually compriselinear or structural motifs and signatures including, but not limitedto, leucine zippers, helix-turn-helix motifs, post-translationalmodification sites such as glycosylation sites, ubiquitination sites,alpha helices, and beta sheets, signal sequences encoding signalpeptides which direct the secretion of the encoded proteins, sequencesimplicated in transcription regulation such as homeoboxes, acidicstretches, enzymatic active sites, substrate binding sites, andenzymatic cleavage sites. Such domains may present a particularbiological activity such as DNA or RNA-binding, secretion of proteins,transcription regulation, enzymatic activity, substrate bindingactivity, etc.

In a preferred embodiment, domains comprise a number of amino acids thatis any integer between 6 and 1000. Domains may be synthesized using anymethods known to those skilled in the art, including those disclosedherein. Methods for determining the amino acids which make up a domainwith a particular biological activity include mutagenesis studies andassays to determine the biological activity to be tested, as well asbioinformatic methods for recognizing domains, motifs, or signatures,e.g., in a database such as Prosite (Hofmann et al., (1999) Nucl. AcidsRes. 27:215-219; Bucher and Bairoch (1994) Proceedings 2nd InternationalConference on Intelligent Systems for Molecular Biology. Altman et al,Eds., pp 53-61, AAAIPress, Menlo Park), Pfam (Sonnhammer, et al., (1997)Proteins. 28(3):405-20; Henikoff et al., (2000) Electrophoresis 21(9):1700-6; Bateman et al., (2000) Nucleic Acids Res. 28(1):263-6), Blocks,Print, Prodom, Sbase, Smart, Dali/FSSP, HSSP, CATH, SCOP, COG. For areview on available databases, see issue 1 of volume 28 of Nucleic AcidResearch (2000).

Epitopes and Antibody Fusions.

A preferred embodiment of the present invention is directed toepitope-bearing polypeptides and epitope-bearing polypeptide fragments.These epitopes may be “antigenic epitopes” or both an “antigenicepitope” and an “immunogenic epitope”. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response in vivowhen the polypeptide is the immunogen. On the other hand, a region ofpolypeptide to which an antibody binds is defined as an “antigenicdeterminant” or “antigenic epitope.” The number of immunogenic epitopesof a protein generally is less than the number of antigenic epitopes(see, e.g., Geysen et al., (1984), PNAS 81:3998-4002). It isparticularly noted that although a particular epitope may not beimmunogenic, it is nonetheless useful since antibodies can be made toboth immunogenic and antigenic epitopes. The present epitopes may belinear (i.e., composed of a contiguous sequence of amino acids repeatedalong the polypeptide chain) or nonlinear (also called “conformational”,i.e., composed of amino acids brought into proximity as a result of thefolding of the polypeptide chain).

An epitope can comprise as few as 3 amino acids in a spatialconformation, which is unique to the epitope. Generally an epitopeconsists of at least 6 such amino acids, and more often at least 8-10such amino acids. In preferred embodiment, antigenic epitopes comprise anumber of amino acids that is any integer between 3 and 50. Fragmentswhich function as epitopes may be produced by any conventional means(see, e.g., Houghten (1985), PNAS 82:5131-5135), also further describedin U.S. Pat. No. 4,631,21. Methods for determining the amino acids whichmake up an epitope include x-ray crystallography, 2-dimensional nuclearmagnetic resonance, and epitope mapping, e.g., the Pepscan methoddescribed by Geysen, et al. (1984); WO 84/03564; and WO 84/03506.Nonlinear epitopes are determined by methods such as proteinfootprinting (U.S. Pat. No. 5,691,448). Another example is the algorithmof Jameson and Wolf, (1988), Comp. Appl. Biosci. 4:181-186. TheJameson-Wolf antigenic analysis, for example, may be performed using thecomputer program PROTEAN, using default parameters (Version 4.0 Windows,DNASTAR, Inc., 1228 South Park Street Madison, Wis.). Epitopes may alsobe identified in vivo by testing for an antigenic response usingstandard methods.

All fragments of the polypeptides of the present invention, at least 6,8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 125, 150, 175, 200,225, 250, 275, or 300 amino acids residues in length, are included inthe present invention as being useful as antigenic linear epitopes.Polypeptides of the present invention that are not specificallydescribed as immunogenic are not considered non-antigenic as they may beantigenic in vivo. The epitope-bearing fragments of the presentinvention preferably comprise 6 to 50 amino acids (i.e. any integerbetween 6 and 50, inclusive) of a polypeptide of the present invention.Any number of epitope-bearing fragments of the present invention mayalso be excluded.

Nonlinear epitopes comprise more than one noncontiguous polypeptidesequence of at least one amino acid each. Such epitopes result fromnoncontiguous polypeptides brought into proximity by secondary,tertiary, or quaternary structural features. Preferred polypeptidesproviding nonlinear epitopes are formed by a contiguous surface ofnatively folded protein and are thus at least 10 amino acids in length,further preferably 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 125,150, 175, 200, 225, 250, 275, or 300 amino acids of a polypeptide of thepresent invention. Additionally, nonlinear epitopes may be formed bysynthetic peptides that mimic an antigenic site or contiguous surfacenormally presented on a protein in the native conformation.

Immunogenic epitopes may be presented together with a carrier protein,such as an albumin, to an animal system (such as rabbit or mouse) or, ifit is long enough (at least about 25 amino acids), without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

Epitope-bearing polypeptides of the present invention are used to induceantibodies according to methods well known in the art including, but notlimited to, in vivo immunization, in vitro immunization, and phagedisplay methods (see, e.g., Sutcliffe, et al., supra; Wilson, et al.,supra, and Bittle, et al., supra). If in vivo immunization is used,animals may be immunized with free peptide; however, anti-peptideantibody titer may be boosted by coupling of the peptide to amacromolecular carrier using standard methods, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. Animals such as rabbits, rats andmice are immunized with either free or carrier-coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 μgs of peptide or carrier protein and Freund'sadjuvant. Several booster injections may be needed, for instance, atintervals of about two weeks, to provide a useful titer of anti-peptideantibody, which can be detected, for example, by ELISA assay using freepeptide adsorbed to a solid surface. The titer of anti-peptideantibodies in serum from an immunized animal may be increased byselection of anti-peptide antibodies, for instance, by adsorption to thepeptide on a solid support and elution of the selected antibodiesaccording to methods well known in the art.

As one of skill in the art will appreciate, and discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to heterologous polypeptide sequences.For example, the polypeptides of the present invention may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, any combination thereof including both entiredomains and portions thereof) resulting in chimeric polypeptides. Thesefusion proteins facilitate purification, and show an increased half-lifein vivo (see, e.g., EPA 0,394,827; and Traunecker et al., (1988),Nature. 331:84-86; Fountoulakis et al., (1995) Biochem. 270:3958-3964).Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, orcodon-shuffling (collectively referred to as “DNA shuffling”; see, forexample, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,834,252; 5,837,458; andPatten, et al. (1997), Curr Opinion Biotechnol. 8:724-733; Harayama(1998), Trends Biotechnol. 16(2): 76-82; Hansson et al., (1999), J. Mol.Biol. 287:265-276; and Lorenzo and Blasco (1998) Biotechniques.24(2):308-313). The present invention further encompasses anycombination of the polypeptide fragments listed in this section.

Antibodies

Definitions

The present invention further relates to antibodies and T-cell antigenreceptors (TCR), which specifically bind the polypeptides, and morespecifically, the epitopes of the polypeptides of the present invention.The antibodies of the present invention include IgG (including IgG1,IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM,and IgY. The term “antibody” (Ab) refers to a polypeptide or group ofpolypeptides which are comprised of at least one binding domain, where abinding domain is formed from the folding of variable domains of anantibody molecule to form three-dimensional binding spaces with aninternal surface shape and charge distribution complementary to thefeatures of an antigenic determinant of an antigen, which allows animmunological reaction with the antigen. As used herein, the term“antibody” is meant to include whole antibodies, including single-chainwhole antibodies, and antigen binding fragments thereof. In a preferredembodiment the antibodies are human antigen binding antibody fragmentsof the present invention include, but are not limited to, Fab, Fab′F(ab)2 and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera V_(L) or V_(H) domain. The antibodies may be from any animal originincluding birds and mammals. Preferably, the antibodies are human,murine, rabbit, goat, guinea pig, camel, horse, or chicken.

Antigen-binding antibody fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentire or partial of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are any combinations of variableregion(s) and hinge region, CH1, CH₂, and CH3 domains. The presentinvention further includes chimeric, humanized, and human monoclonal andpolyclonal antibodies, which specifically bind the polypeptides of thepresent invention. The present invention further includes antibodiesthat are anti-idiotypic to the antibodies of the present invention.

The antibodies of the present invention may be monospecific, bispecific,and trispecific or have greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for heterologous compositions, such as aheterologous polypeptide or solid support material. See, e.g., WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., (1991),J. Immunol. 147:60-69; U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819,4,714,681, 4,925,648; Kostelny et al., (1992), J. Immunol.148:1547-1553.

Antibodies of the present invention may be described or specified interms of the epitope(s) or epitope-bearing portion(s) of a polypeptideof the present invention, which are recognized or specifically bound bythe antibody. The antibodies may specifically bind a complete proteinencoded by a nucleic acid of the present invention, or a fragmentthereof. Antibodies which specifically bind any epitope or polypeptideof the present invention may also be excluded as individual species.

Thus, another embodiment of the present invention is a purified orisolated antibody capable of specifically binding to any of thepolypeptides of the present invention. In one aspect of this embodiment,the antibody is capable of binding to a linear epitope-containingpolypeptide comprising at least 6 consecutive amino acids, preferably atleast 8 to 10 consecutive amino acids, more preferably at least 12, 15,20, 25, 30, 40, 50, or 100 consecutive amino acids of a polypeptides ofthe present invention. In another aspect of this embodiment, theantibody is capable of binding to a nonlinear epitope-containingpolypeptide comprising 10 amino acids in length, further preferably 12,15, 20, 25, 30, 35, 40, 50, 60, 75, or 100 amino acids, furtherpreferably, a contiguous surface of the native conformation of apolypeptide of the present application.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not specificallybind any other analog, ortholog, or homolog of the polypeptides of thepresent invention are included. Antibodies that do not bind polypeptideswith less than 95%, less than 90%, less than 85%, less than 80%, lessthan 75%, less than 70%, less than 65%, less than 60%, less than 55%,and less than 50% identity (as calculated using methods known in the artand described herein, e.g., using FASTDB and the parameters set forthherein) to a polypeptide of the present invention are also included inthe present invention. Further included in the present invention areantibodies, which only bind polypeptides encoded by polynucleotides,which hybridize to a polynucleotide of the present invention understringent hybridization conditions (as described herein). Antibodies ofthe present invention may also be described or specified in terms oftheir binding affinity. Preferred binding affinities include those witha dissociation constant or Kd less than 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M,5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M,5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, and10⁻⁵M.

The invention also concerns a purified or isolated antibody capable ofspecifically binding to a mutated GENSET protein or to a fragment orvariant thereof comprising an epitope of the mutated GENSET protein.

Preparation of Antibodies

The antibodies of the present invention may be prepared by any suitablemethod known in the art. For example, a polypeptide of the presentinvention or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing “polyclonalantibodies”. As used herein, the term “monoclonal antibody” is notlimited to antibodies produced through hybridoma technology but itrather refers to an antibody that is derived from a single clone,including eukaryotic, prokaryotic, or phage clone, and not the method bywhich it is produced. Monoclonal antibodies can be prepared using a widevariety of techniques known in the art including the use of hybridoma,recombinant, and phage display technology.

Hybridoma techniques include those known in the art (see, e.g., Harlowand Lane, (1988) Antibodies A Laboratory Manual. Cold Spring HarborLaboratory. pp. 53-242; Hammerling (1981), Monoclonal Antibodies andT-Cell Hybridomas, Elsevier, N.Y. 563-681; Kohler and Milstein, (1975)Nature 256:495; Davis, et al., Basic Methods in Molecular Biology, ed.,Elsevier Press, NY (1986), Section 21-2).).

Briefly, a mouse is repetitively inoculated with a few micrograms of theGENSET protein, or a portion thereof, over a period of a few weeks. Themouse is then sacrificed, and the antibody producing cells of the spleenisolated. The spleen cells are fused by means of polyethylene glycolwith mouse myeloma cells, and antibody-producing clones are identified(see, e.g., Engvall, (1980) Meth. Enzymol. 70:419). Selected positiveclones can be expanded and their monoclonal antibody product harvestedfor use.

Further, Fab and F(ab′)2 fragments may be produced, for example, fromhybridoma-produced antibodies by proteolytic cleavage, using enzymessuch as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2fragments Polyclonal antiserum containing antibodies to heterogeneousepitopes in the GENSET protein or a portion thereof can be prepared byimmunizing suitable non-human animals (e.g., mouse, rat, rabbit, goat,or horse) with the GENSET protein or a portion thereof, which can beunmodified or modified to enhance immunogenicity (see, e.g., Vaitukaitiset al., (1971) J. Clin. Endocrinol. Metab. 33:988-991; Ouchterlony etal., (1973) Chap. 19 in: Handbook of Experimental Immunology D. Wier(ed) Blackwell). The protein or fragment is typically introduced intothe non-human mammal in the presence of an appropriate adjuvant (e.g.aluminum hydroxide, RIBI, etc.).

Serum from the immunized animal is collected, treated and testedaccording to known procedures. If the serum contains polyclonalantibodies to undesired epitopes, the polyclonal antibodies can bepurified by immunoaffinity chromatography. Affinity of the antisera forthe antigen is determined using standard methods.

Alternatively, antibodies of the present invention can be producedthrough the application of recombinant DNA technology or throughsynthetic chemistry using methods known in the art. In phage displaymethods, for example, functional antibody domains are displayed on thesurface of a phage particle, which carries polynucleotide sequencesencoding them, and phage with a desired binding property are selectedfrom a repertoire or combinatorial antibody library (e.g. human ormurine) by selecting directly with antigen, typically antigen bound orcaptured to a solid surface or bead (see, e.g., Brinkman et al., (1995)J. Immunol. Methods, 182:41-50; Ames et al., (1995), J. Immunol. Meth.,184:177-186; Kettleborough et al., (1994), Eur. L Immunol., 24:952-958;Persic et al., (1997), Gene, 1879-81; Burton et al. (1994), Adv.Immunol., 57:191-280; PCT/GB91/01134; WO 90/02809; WO 91/10737; WO92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S.Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908,5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225,5,658,727 and 5,733,743). After phage selection, the antibody codingregions from the phage can be isolated and used to generate wholeantibodies, including human antibodies, or any other desired antigenbinding fragment, and expressed in any desired host including mammaliancells, insect cells, plant cells, yeast, and bacteria (see, e.g., WO92/22324; Mullinax et al., (1992), BioTechniques. 12(6):864-869; andSawai et al., (1995), AJRI 34:26-34; and Better et al., (1988), Science.240:1041-1043).

Further teaching regarding the preparation of single-chain Fvs andantibodies is provided in U.S. Pat. Nos. 4,946,778 and 5,258,498; Hustonet al., (1991), Meth. Enymol. 203:46_(—)88; Shu, et al., (1993), PNAS90:7995-7999; and Skerra, et al., (1988), and Science 240:1038-1040. Forsome uses, including in vivo use of antibodies in humans and in vitrodetection assays, it may be preferable to use chimeric, shuffled,humanized, or human antibodies (see e.g., Morrison, (1985); Oi et al.,(1986), BioTechniques 4:214; Gillies et al., (1989), J. Immunol.Methods. 125:191-202; and U.S. Pat. No. 5,807,715; EP 0 239 400; WO91/09967; U.S. Pat. Nos. 5,530,101; and 5,585,089, EP 0 592 106; EP 0519 596; Padlan (1991), Molec. Immunol. 28(4/5):489-498; Studnicka etal., (1994), Protein Engineering. 7(6):805-814; Roguska et al., (1994),PNAS 91:969-973, U.S. Pat. No. 5,565,332, U.S. Pat. Nos. 4,444,887,4,716,111, 5,545,806, and 5,814,318; WO 98/46645; WO 98/50433; WO98/24893; WO 96/34096; WO 96/33735; and WO 91/10741).

Further included in the present invention are antibodies recombinantlyfused or chemically conjugated (including both covalent and non-covalentconjugations) to any heterologous molecule, such as a polypeptide of thepresent invention, another polypeptide, a label useful for detectionassays, or an effector molecule such as a drug or toxin (see, e.g., WO92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 0396 387). Fused antibodies may also be used to target the polypeptidesof the present invention to particular cell types, either in vitro or invivo, by fusing or conjugating the polypeptides of the present inventionto antibodies specific for particular cell surface receptors, and mayalso be used in vitro immunoassays and purification methods usingmethods known in the art (see e.g., Harbor, et al. supra; WO 93/21232;EP 0 439 095; Naramura et al., (1994), Immunol. Lett. 39:91-99; U.S.Pat. No. 5,474,981; Gillies et al., (1992), PNAS 89:1428-1432; Fell etal., (1991), J. Immunol. 146:2446-2452).

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof, and/or the hinge region, CH1 domain, CH2domain, or CH3 domain or portions thereof, as well as to portions of IgAor IgM. Methods for fusing or conjugating the polypeptides of thepresent invention to antibody portions are known in the art. See e.g.,U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851,5,112,946; EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570;Ashkenazi et al., (1991), PNAS 88:10535-10539; Zheng, et al. (1995), J.Immunol. 154:5590-5600; and Vil, et al. (1992), PNAS 89:11337-11341.

Non-human animals or mammals, whether wild-type or transgenic, whichexpress a different species of GENSET polypeptide than the one to whichantibody binding is desired, and animals which do not express a GENSETpolypeptide (i.e. a GENSET knock out animal as described herein) areparticularly useful for preparing antibodies, as such animals willrecognize all or most of the exposed regions of a GENSET protein asforeign antigens, and therefore produce antibodies with a wider array ofGENSET epitopes. Moreover, smaller polypeptides with only 10 to 30 aminoacids may be useful in obtaining specific binding to any one of theGENSET proteins. In addition, the humoral immune system of animals whichproduce a species of GENSET that resembles the antigenic sequence willpreferentially recognize the differences between the animal's nativeGENSET species and the antigen sequence, and produce antibodies to theseunique sites in the antigen sequence. Such a technique will beparticularly useful in obtaining antibodies that specifically bind toany one of the GENSET proteins.

A preferred embodiment of the invention is a method of specificallybinding an antibody or antibody fragment to a GENSET polypeptide. Thismethod comprises the step of contacting a GENSET polypeptide-specificantibody or fragment thereof with a GENSET polypeptide underantibody-binding conditions. Further included is a method ofspecifically binding an antibody or antibody fragment to an epitope,domain, or fragment of a GENSET polypeptide. This method may be used to,for example, detect, purify, or modify the activity of GENSETpolypeptides, as discussed herein.

Antibodies of the invention can be used to assay protein levels in atest sample or biological sample using methods known to those of skillin the art. Antibody-based methods useful for detecting protein includeimmunoassays, such as the enzyme linked immunosorbent assay (ELISA) andradioimimunoassay (RIA). Suitable antibody assay labels are known in theart and include enzyme labels, such as glucose oxidase, horseradishperoxidase, and alkaline phosphatase; radioisotopes, such as iodine(125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In),and technetium (99Tc); luminescent labels, such luminol, isolumino,theromatic acridinium ester, imidazole, acridinium salt, oxalate ester,luciferin, luciferase, and aequorin; and fluorescent labels, such asfluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde, and fluorescamine.

Uses of Polynucleotides

Uses of Polynucleotides as Reagents

The polynucleotides of the present invention may be used as reagents inisolation procedures, diagnostic assays, and forensic procedures. Forexample, sequences from the GENSET polynucleotides of the invention maybe detectably labeled and used as probes to isolate other sequencescapable of hybridizing to them. In addition, sequences from the GENSETpolynucleotides of the invention may be used to design PCR primers to beused in isolation, diagnostic, or forensic procedures.

To Find Corresponding Genomic DNA Sequences

The GENSET cDNAs of the invention may also be used to clone sequenceslocated in the vicinity, preferably upstream of the cDNAs of theinvention on the corresponding genomic DNA. Such sequences may becapable of regulating gene expression, including promoter sequences,enhancer sequences, and other sequences which influence transcription ortranslation levels.

Use of cDNAs or Fragments Thereof to Clone Upstream Sequences fromGenomic DNA

Sequences derived from polynucleotides of the inventions may be used toisolate the promoters of the corresponding genes using chromosomewalking techniques (e.g., using the GenomeWalker™ kit from Clontech).Once the upstream genomic sequences have been cloned and sequenced,prospective promoters and transcription start sites within the upstreamsequences may be identified by comparing the sequences upstream of thepolynucleotides of the inventions with databases containing knowntranscription start sites, transcription factor binding sites, orpromoter sequences.

In addition, promoters in the upstream sequences may be identified usingpromoter reporter vectors, e.g., by placing a reporter gene (e.g.,secreted alkaline phosphatase, luciferase, beta-galactosidase, or greenfluorescent protein) under the control of regulatory activepolynucleotide fragments or variants of the GENSET promoter regionlocated upstream of the first exon of the GENSET gene. A large number ofsuitable promoter reporter vectors are known in the art. The promotersand other regulatory sequences located upstream of the polynucleotidesof the inventions may be used to design expression vectors capable ofdirecting the expression of an inserted gene in a desired spatial,temporal, developmental, or quantitative manner.

To Find Similar Sequences

Polynucleotides of the invention may be used to isolate and/or purifynucleic acids similar thereto using any methods well known to thoseskilled in the art including the techniques based on hybridization or onamplification described in this section. These methods may be used toobtain the genomic DNAs which encode the mRNAs from which the GENSETcDNAs are derived, mRNAs corresponding to GENSET cDNAs, or nucleic acidswhich are homologous to GENSET cDNAs or fragments thereof, such asvariants, species homologues or orthologs.

Hybridization-Based Methods

Techniques for identifying cDNA clones in a cDNA library which hybridizeto a given probe sequence are disclosed in Sambrook et al., (1989)Molecular Cloning: A Laboratory Manual. (2ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.), and in Hames and Higgins (1985)Nucleic Acid Hybridization: A Practical Approach (Hames and Higgins Ed.,IRL Press, Oxford). The same techniques may be used to isolate genomicDNAs.

A probe comprising at least 10 consecutive nucleotides from a GENSETcDNA or fragment thereof, is labeled using standard methods with adetectable label such as a radioisotope or a fluorescent molecule. ThecDNAs or genomic DNAs in the library are transferred to a nitrocelluloseor nylon filter and denatured. After blocking of nonspecific sites, thefilter is incubated with the labeled probe for an amount of timesufficient to allow binding of the probe to cDNAs or genomic DNAscontaining a sequence capable of hybridizing thereto.

By varying the stringency of the hybridization conditions used toidentify cDNAs or genomic DNAs which hybridize to the detectable probe,cDNAs or genomic DNAs having different levels of identity to the probecan be identified and isolated as described below.

Stringent Conditions

“Stringent hybridization conditions” are defined as conditions in whichonly nucleic acids having a high level of identity to the probe are ableto hybridize to said probe. These conditions may be calculated asfollows:

For probes between 14 and 70 nucleotides in length the meltingtemperature (Tm) is calculated using the formula:Tm=81.5+16.6(log(Na+))+0.41(fraction G+C)−(600/N) where N is the lengthof the probe.

If the hybridization is carried out in a solution containing formamide,the melting temperature may be calculated using the equation:Tm=81.5+16.6(log(Na+))+0.41(fraction G+C)−(0.63% formamide)−(600/N)where N is the length of the probe.

Prehybridization may be carried out in 6×SSC, 5×Denhardt's reagent, 0.5%SDS, 100 μg denatured fragmented salmon sperm DNA or 6×SSC, 5×Denhardt'sreagent, 0.5% SDS, 100 μg denatured fragmented salmon sperm DNA, 50%formamide (see, e.g., Sambrook et al., 1986).

Hybridization is conducted according to standard methods. For probesover 200 nucleotides in length, the hybridization may be carried out at15-25° C. below the Tm. For shorter probes, such as oligonucleotideprobes, the hybridization may be conducted at 15-25° C. below the Tm.Preferably, for hybridizations in 6×SSC, the hybridization is conductedat approximately 68° C. Preferably, for hybridizations in 50% formamidecontaining solutions, the hybridization is conducted at approximately42° C.

Following hybridization, the filter is washed in 2×SSC, 0.1% SDS at roomtemperature for 15 minutes. The filter is then washed with 0.1×SSC, 0.5%SDS at room temperature for 30 minutes to 1 hour. Thereafter, thesolution is washed at the hybridization temperature in 0.1×SSC, 0.5%SDS. A final wash is conducted in 0.1×SSC at room temperature.

Nucleic acids which have hybridized to the probe are identified byautoradiography or other conventional techniques.

Low and Moderate Conditions

Changes in the stringency of hybridization and signal detection areprimarily accomplished through the manipulation of formamideconcentration (lower percentages of formamide result in loweredstringency); salt conditions, or temperature. The above procedure maythus be modified to identify nucleic acids having decreasing levels ofidentity to the probe sequence. For example, the hybridizationtemperature may be decreased in increments of 5° C. from 68° C. to 42°C. in a hybridization buffer having a sodium concentration ofapproximately 1M. Following hybridization, the filter may be washed with2×SSC, 0.5% SDS at the temperature of hybridization. These conditionsare considered to be “moderate” conditions above 50° C. and “low”conditions below 50° C. Alternatively, the hybridization may be carriedout in buffers, such as 6×SSC, containing formamide at a temperature of42° C. In this case, the concentration of formamide in the hybridizationbuffer may be reduced in 5% increments from 50% to 0% to identify cloneshaving decreasing levels of identity to the probe. Followinghybridization, the filter may be washed with 6×SSC, 0.5% SDS at 50° C.These conditions are considered to be “moderate” conditions above 25%formamide and “low” conditions below 25% formamide. cDNAs or genomicDNAs which have hybridized to the probe are identified byautoradiography or other conventional techniques.

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents (e.g.Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, etc.)used to suppress background in hybridization experiments.

PCR-Based Methods

In addition to the above described methods, other protocols areavailable to obtain homologous cDNAs using GENSET cDNA of the presentinvention or fragment thereof, as described below.

cDNAs may be prepared by obtaining mRNA from the tissue, cell, ororganism of interest, e.g., using mRNA preparation procedures utilizingpolyA selection procedures or other techniques known to those skilled inthe art. A first primer capable of hybridizing to the polyA tail of themRNA (e.g. an oligo(T) primer) is hybridized to the mRNA, and a reversetranscription reaction is performed to generate a first cDNA strand(see, e.g., Current Protocols in Molecular Biology, John Wiley and Sons,Inc. 1997 and Sambrook, et al., 1989). Typically, such oligo(T) primerscomprise an additional sequence upstream of the poly(dT) stretch whichfacilitates subsequent manipulation of the DNA, such as a restrictionsite-containing sequence.

The first cDNA strand is then hybridized to a second primer containingat least 10 consecutive nucleotides of a polynucleotide of theinvention. Often, the second primer used contains sequences locatedupstream of the translation initiation site. The second primer isextended to generate a second cDNA strand complementary to the firstcDNA strand. Alternatively, RT-PCR may be performed as described aboveusing primers from both ends of the cDNA to be obtained. See, e.g.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc. 1997 andSambrook, et al., 1989.

Other Protocols

Alternatively, other procedures may be used for obtaining homologouscDNAs. In one approach, cDNAs are prepared from mRNA and cloned intodouble stranded phagemids using standard methods. The cDNA library inthe double stranded phagemids is then rendered single stranded, abiotinylated oligonucleotide comprising the sequence of a fragment of aknown GENSET cDNA, genomic DNA or fragment thereof is hybridized to thesingle stranded phagemids. Hybrids between the biotinylatedoligonucleotide and phagemids are isolated and the correspondingphagemids are released from the beads and converted into double strandedDNA using a primer specific for the GENSET cDNA or fragment used todesign the biotinylated oligonucleotide. Alternatively, protocols suchas the Gene Trapper kit (Gibco BRL), may be used.

As a Chromosome Marker

GENSET polynucleotides may be mapped to their chromosomal locationsusing any methods or techniques known to those skilled in the artincluding radiation hybrid (RH) mapping (See, e.g., Benham et al. (1989)Genomics 4:509-517 and Cox et al., (1990) Science 250:245-250; andSchuler et al., (1996) Science 274:540-546), PCR-based mapping, andFluorescence in situ hybridization (FISH) mapping, as described below.

Mapping of cDNAs to Human Chromosomes Using PCR Techniques GENSET cDNAsand genomic DNAs may be assigned to human chromosomes using PCR basedmethodologies. In such approaches, oligonucleotide primer pairs aredesigned from the cDNA sequence, and PCR is used to screen a series ofsomatic cell hybrid cell lines containing defined sets of humanchromosomes. Only those somatic cell hybrids with chromosomes containingthe human gene corresponding to the GENSET cDNA or genomic DNA willyield an amplified fragment, and the single human chromosome present inall cell hybrids that give rise to an amplified fragment is thechromosome containing that GENSET cDNA or genomic DNA (see, e.g.,Ledbetter et al., (1990) Genomics 6:475-481).

Mapping of cDNAs to Chromosomes Using Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) allows the GENSET cDNA orgenomic DNA to be mapped to a particular location on a given chromosome.The chromosomes to be used for fluorescence in situ hybridizationtechniques may be obtained from a variety of sources including cellcultures, tissues, or whole blood (see, e.g., Chemf et al., (1990) PNAS87:6639-6643).

Use of cDNAs to Construct or Expand Chromosome Maps

Once the GENSET cDNAs or genomic DNAs have been assigned to particularchromosomes, they may be utilized to construct a high resolution map ofthe chromosomes on which they are located or to identify the chromosomesin a sample.

Chromosome mapping involves assigning a given unique sequence to aparticular chromosome as described above. Once the unique sequence hasbeen mapped to a given chromosome, it is ordered relative to otherunique sequences located on the same chromosome (see, e.g., Nagaraja etal., (1997) Genome Res. 1997 March; 7(3):210-22).

Identification of Genes Associated with Hereditary Diseases or DrugResponse

In another embodiment, any particular GENSET cDNA or genomic DNA may beused as a test probe to associate that GENSET cDNA or genomic DNA with aparticular phenotypic characteristic.

In one embodiment, GENSET cDNAs or genomic DNAs are mapped to aparticular location on a human chromosome using standard techniques andthe location is searched in Mendelian Inheritance in Man (V. McKusick,Mendelian Inheritance in Man; available on line through Johns HopkinsUniversity Welch Medical Library). Often, this search reveals the regionof the human chromosome which contains the GENSET cDNA or genomic DNA tobe a very gene rich region containing several known genes and severaldiseases or phenotypes for which genes have not been identified. Thegene corresponding to this GENSET cDNA or genomic DNA thus becomes animmediate candidate for each of these genetic diseases.

Genomic DNA, mRNA or cDNA from patients with these diseases orphenotypes may then be screened, e.g. using PCR primers from the GENSETcDNA or genomic DNA, or by sequencing, for differences in theexpression, size, or sequence of the GENSET cDNA or genomic DNAs inpatients relative to in disease-free individuals. Any detecteddifference indicates a role for the GENSET gene in the disease orphenotype.

Uses of Polynucleotides in Recombinant Vectors

The present invention also relates to recombinant vectors including theisolated polynucleotides of the present invention, and to host cellsrecombinant for a polynucleotide of the invention, such as the abovevectors, as well as to methods of making such vectors and host cells andfor using them for production of GENSET polypeptides by recombinanttechniques.

Recombinant Vectors

The term “vector” is used herein to designate either a circular or alinear DNA or RNA molecule, which is either double-stranded orsingle-stranded, and which comprises at least one polynucleotide ofinterest that is sought to be transferred in a cell host or in aunicellular or multicellular host organism.

In one preferred embodiment, the present invention provides expressionvectors comprising either a regulatory polynucleotide or a codingnucleic acid of the invention, or both. Expression vectors may be usedto express a GENSET polypeptide for purification, as well as forconstructing transgenic animals and also for gene therapy (including invivo and ex vivo methods). The encoded protein may be transientlyexpressed in a host organism or cell or stably expressed in the hostorganism or cell. The encoded protein may have any of the activitiesdescribed herein. The encoded protein may be a protein which the hostorganism lacks or, alternatively, the encoded protein may augment theexisting levels of the protein in the host organism.

Some of the elements which can be found in the vectors of the presentinvention are described in further detail in the following sections.

General Features of the Expression Vectors of the Invention

Typical expression vectors of the present invention comprise atranscriptional unit comprising an assembly of a genetic element orelements having a regulatory role in gene expression, for example apromoter and a structural or coding sequence which is transcribed intomRNA and eventually translated into a polypeptide, said structural orcoding sequence being operably linked to the regulatory elementsdescribed above, and appropriate transcription initiation andtermination sequences. Additional features may include enhancers, aleader sequence enabling extracellular secretion of translated proteinby a host cell, origins of replication, selectable markers permittingtransformation of the host cell, ribosome binding sites, polyadenylationsignals, splice donor and acceptor sites, transcriptional terminationsequences, and 5′-flanking non-transcribed sequences.

Regulatory Elements

The suitable promoter regions used in the expression vectors accordingto the present invention are chosen taking into account the cell host inwhich the heterologous gene has to be expressed.

A suitable promoter may be heterologous with respect to the nucleic acidfor which it controls the expression or alternatively can be endogenousto the native polynucleotide containing the coding sequence to beexpressed. Promoter regions can be selected from any desired gene using,for example, CAT (chloramphenicol transferase) vectors and morepreferably pKK232-8 and pCM7 vectors. Preferred bacterial promoters arethe LacI, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, thegpt, lambda PR, PL and trp promoters (EP 0036776), the polyhedrinpromoter, or the p10 protein promoter from baculovirus (Kit Novagen)(Smith et al., (1983) Mol. Cell. Biol. 3:2156-2165; O'Reilly et al.(1992), “Baculovirus Expression Vectors: A Laboratory Manual”, W.H.Freeman and Co., New York), the lambda PR promoter or also the trcpromoter.

Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-L.Selection of a convenient vector and promoter is well within the levelof ordinary skill in the art.

Selectable Markers

Selectable markers confer an identifiable change to the cell permittingeasy identification of cells containing the expression construct. Theselectable marker genes for selection of transformed host cells arepreferably dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, TRP1 for S. cerevisiae or tetracycline, rifampicin orampicillin resistance in E. Coli, or levan saccharase for mycobacteria,this latter marker being a negative selection marker.

Preferred Vectors

The present recombinant vectors may be any sort of vector, including,but not limited to, YACs (Yeast Artificial Chromosome), BACs (BacterialArtificial Chromosome), phage, phagemids, cosmids, plasmids, and linearDNA.

Bacterial Vectors

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and a bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of pBR322 (ATCC 37017). Such commercialvectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden),pGEM1 (Promega Biotec, Madison, Wis., USA), pQE70, pQE60, pQE-9(Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A,pNH 16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG(Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).

Bacteriophage Vectors

The P1 bacteriophage vector may contain large inserts ranging from about80 to about 100 kb. The construction of P1 bacteriophage vectors is wellknown in the art (see, e.g., Sternberg (1992) Trends Genet. 8:1-16,Sternberg (1994) Mamm. Genome. 5:397-404, Linton et al., (1993) J. Clin.Invest. 92:3029-3037, McCormick et al., (1994) Genet. Anal. Tech. Appl.11:158-164.

Viral Vectors

Any viral vector can be used to carry out the herein-described methods.In one specific embodiment, the vector is derived from an adenovirus,e.g., human adenovirus type 2 or 5 (see, e.g., Feldman et al. (1996)Medecine/Sciences, 12:47-55, Ohno et al., (1994) Science 265:781-784,French patent application No. FR-93.05954).

Adeno-associated viral vectors are also preferred for theherein-described methods.

Particularly preferred retroviruses for the preparation or constructionof retroviral in vitro or in vitro gene delivery vehicles of the presentinvention include retroviruses selected from the group consisting ofMink-Cell Focus Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis virus and Rous Sarcoma virus. Particularlypreferred Murine Leukemia Viruses include the 4070A and the 1504Aviruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCCNo VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus(ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferredRous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657,VR-726, VR-659 and VR-728). Other preferred retroviral vectors are thosedescribed in Roth et al., (1996) Nature Medicine, 2(9):985-991, WO93/25234, WO 94/06920, Roux et al. (1989), PNAS 86:9079-9083, Julan etal. (1992), J. Gen. Virol. 73:3251-3255, and Neda et al. (1991), J.Biol. Chem. 266:14143-14146.

BAC vectors

The bacterial artificial chromosome (BAC) cloning system (Shizuya et al.(1992), PNAS 89:8794-8797), has been developed to stably maintain largefragments of genomic DNA (100-300 kb) in E. coli. A preferred BAC vectorcomprises a pBeloBAC11 vector that has been described by Kim U-J. et al.(1996), Genomics 34:213-218. BAC vectors, and the construction thereof,are well known in the art, and any suitable

Baculovirus

Another specific suitable host vector system is the pVL1392/1393baculovirus transfer vector (Pharmingen) that is used to transfect theSF9 cell line (ATCC No. CRL 1711) which is derived from Spodopterafrugiperda. Other suitable vectors for the expression of the GENSETpolypeptide of the present invention in a baculovirus expression systeminclude those described by Chai et al. (1993), Biotechnol. Appl.Biochem. 18:259-273; Vlasak, et al. (1983), Eur. J. Biochem.135:123-126, and Lenhard et al., (1996) Gene. 169:187-190.

Delivery of the Recombinant Vectors

To effect expression of the polynucleotides and polynucleotideconstructs of the invention, the constructs must be delivered into acell. This delivery may be accomplished in vitro, as in laboratoryprocedures for transforming cell lines, or in vivo or ex vivo, as in thetreatment of certain diseases states.

One mechanism is viral infection, where the expression construct isencapsulated in an infectious viral particle (see, U.S. Pat. No.5,968,821). The expression construct, preferably a recombinant viralvector as discussed herein, is used to transduce packaging cells, whichthen produce infectious viral particles including the expressionconstruct. The particles are then used to transduce eukaryotic cells(see, Miller, A. D. (1990) Blood 76:271; U.S. Pat. No. 6,228,844).

Replication defective retrovirus comprising a GENSET polynucleotide maybe packaged into virions, which can then be used to infect a target cellthrough the use of a helper virus by standard techniques (see, e.g.,Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.)Greene Publishing Associates, (1989)). Any of a large number ofretroviruses can be used and are well known in the art (see, e.g.,Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988)PNAS 85:6460-6464; Wilson, et al. (1988) PNAS 85:3014-3018; Armentano,et al. (1990) PNAS 87:6141-6145; Huber, et al. (1991) PNAS 88:8039-8043;Ferry, et al. (1991) PNAS 88:8377-8381; Chowdhury, et al. (1991) Science254:1802-1805; van Beusechem, et al. (1992) PNAS 89:7640-7644; Kay, etal. (1992) Human Gene Therapy 3:641-647; Dai, et al. (1992) PNAS89:10892-10895; Hwu, et al. (1993) J. Immunol. 150:4104-4115).

Another viral gene delivery system useful in the present inventionutilizes adenovirus-derived vectors. The genome of an adenovirus can bemanipulated such that it encodes a gene product of interest, but isinactivated in terms of its ability to replicate in a normal lytic virallife cycle (see, e.g., Berkner, et al. (1988) BioTechniques 6:616;Rosenfeld, et al. (1991) Science 252:431-434; and Rosenfeld, et al.(1992) Cell 68:143-155). Any standard adenoviral vector may be used inthe present invention (see, e.g., Jones, et al. (1979) Cell 16:683;Graham, et al. in Methods in Molecular Biology, E. J. Murray, Ed.(Humana, Clifton, N. J., 1991) vol. 7. pp. 109-127).

Yet another viral vector system useful for delivery of polynucleotidesis the adeno-associated virus (AAV). Adeno-associated virus is anaturally occurring defective virus that requires another virus, such asan adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle (see Muzyczka, et al., Curr.Top. Micro. Immunol. (1992) 158:97-129). Any standard AAV vector may beused in the present invention (see, e.g., Flotte et al., (1992) Am. J.Respir. Cell Mol. Biol. 7:349-356; Am. J. Respir. Cell. Mol. Biol.7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlinet al. (1989) J. Virol. 62:1963-1973, Tratschin, et al. (1985) Mol.Cell. Biol. 5:3251-3260, Hermonat, et al. (1984) PNAS 81:6466-6470;Tratschin, et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford, etal. (1988) Mol. Endocrinol. 2:32-39; Tratschin, et al. (1984) J. Virol.51:611-619; and Flotte, et al. (1993) J. Biol. Chem. 268:3781-3790).

Other viral vector systems that may have application in gene therapyhave been derived from herpes virus, vaccinia virus, and several RNAviruses. In particular, herpes virus vectors may provide a uniquestrategy for persistence of inserted gene expression in cells of thecentral nervous system and ocular tissue (Pepose, et al. (1994) InvestOpthalmol V is Sci 35:2662-2666).

Several non-viral methods for the transfer of polynucleotides intocells, e.g., mammalian cells, in vivo, in vitro, or ex vivo, are alsocontemplated by the present invention, and include, without beinglimited to, calcium phosphate precipitation (Graham et al., (1973)Virol. 52:456-457; Chen et al. (1987) Mol. Cell. Biol. 7:2745-2752);DEAE-dextran (Gopal (1985) Mol. Cell. Biol., 5:1188-1190);electroporation (Tur-Kaspa et al. (1986) Mol. Cell. Biol. 6:716-718;Potter et al., (1984) PNAS 81(22):7161-7165); direct microinjection(Harland et al., (1985) J. Cell. Biol. 101: 1094-1095); DNA-loadedliposomes (Nicolau et al., (1982) Biochim. Biophys. Acta. 721:185-190;Fraley et al., (1979) PNAS 76:3348-3352); and receptor-mediatedtransfection. (Wu and Wu (1987), J. Biol. Chem. 262:4429-4432; and Wuand Wu (1988), Biochemistry 27:887-892).

One specific embodiment for a method for delivering a protein or peptideto the interior of a cell of a vertebrate in vivo or in vitro comprisesthe step of introducing a preparation comprising a physiologicallyacceptable carrier and a naked polynucleotide operatively coding for thepolypeptide of interest into the interstitial space of a tissuecomprising the cell, whereby the naked polynucleotide is taken up intothe interior of the cell and has a physiological effect (see, e.g., WO90/11092, WO 95/11307, Tascon et al. (1996), Nature Medicine2(8):888-892, and Huygen et al., (1996) Nature Medicine 2(8):893-898).Naked polynucleotides of the invention may also be introduced into cellsusing particle bombardment (biolistic), e.g., DNA-coatedmicroprojectiles accelerated to a high velocity allowing them to piercecell membranes and enter cells without killing them (Klein et al.,(1987) Nature 327:70-73).

Liposomal preparations for use in the present invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations. However, cationic liposomes are particularly preferredbecause a tight charge complex can be formed between the cationicliposome and the polyanionic nucleic acid. Cationic liposomes have beenshown to mediate intracellular delivery of plasmid DNA (Felgner, et al.,PNAS (1987) 84:7413-7416); mRNA (Malone, et al., PNAS (1989)86:6077-6081); and purified transcription factors (Debs et al., J. Biol.Chem. (1990) 265:10189-10192), in functional form. Any of a large numberof cationic liposomes can be used.N[1-2,3-dioleyloxy)propyl-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. Other commercially availableliposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Similarly, anionic and neutral liposomes are readily available, such asfrom AvantiPolar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphosphatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art, as are various combinations of liposomes.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art (Straubinger, et al.,Methods of Immunology (1983), 101:512-527; U.S. Pat. No. 5,965,421).Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10 (with about 5:1 to about 1:5 or about 3:1 to about 1:3 beingpreferred). Additionally, liposomes may be targeted to specific celltypes by embedding a targeting moiety such as a member of areceptor-receptor ligand pair into the lipid envelope of the vesicle(see, e.g., U.S. Pat. No. 6,177,433, U.S. Pat. No. 6,110,490, and P.C.TNo. WO9704748).

The amount of vector to be injected to the desired host organism variesaccording to the site of injection. As an indicative dose, it will beinjected between 0.1 and 100 μg of the vector in an animal body,preferably a mammal body, for example a mouse body.

Secretion Vectors

Some of the GENSET cDNAs or genomic DNAs of the invention may also beused to construct secretion vectors capable of directing the secretionof the proteins encoded by genes inserted in the vectors. Such secretionvectors may facilitate the purification or enrichment of the proteinsencoded by genes inserted therein by reducing the number of backgroundproteins from which the desired protein must be purified or enriched.Exemplary secretion vectors are described below.

The secretion vectors of the present invention comprise a promotercapable of directing gene expression in the host cell, tissue, ororganism of interest, a cloning site for inserting a coding sequence,and a signal sequence from a polynucleotide of the invention is operablylinked to the promoter such that the mRNA transcribed from the promoterwill direct the translation of the signal peptide. The host cell,tissue, or organism may be any cell, tissue, or organism whichrecognizes the signal peptide encoded by the signal sequence in theGENSET cDNA or genomic DNA. Suitable hosts include mammalian cells,tissues or organisms, avian cells, tissues, or organisms, insect cells,tissues or organisms, or yeast. The signal sequences may also beinserted into vectors designed for gene therapy.

The secretion vector may be DNA or RNA and may integrate into thechromosome of the host, be stably maintained as an extrachromosomalreplicon in the host, be an artificial chromosome, or be transientlypresent in the host. Preferably, the secretion vector is maintained inmultiple copies in each host cell.

Cell Hosts

Another object of the invention comprises a host cell that has beentransformed or transfected with one of the polynucleotides describedherein, and in particular a polynucleotide either comprising a GENSETpolypeptide-encoding polynucleotide regulatory sequence or thepolynucleotide coding for a GENSET polypeptide. Also included are hostcells that are transformed (prokaryotic cells), transfected (eukaryoticcells), or transduced with a recombinant vector such as one of thosedescribed above. Preferred host cells used as recipients for theexpression vectors of the invention include prokaryotic host cells suchas Escherichia coli strains (I.E.DH5-α strain), Bacillus subtilis,Salmonella typhimurium, and strains from species like Pseudomonas,Streptomyces and Staphylococcus, as well as eukaryotic host cells suchas HeLa cells, Cv 1 cells, COS cells, Sf-9 cells, C127 cells, 3T3, CHO,human kidney 293, and BHK cells.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with the polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous polynucleotides (see,e.g., U.S. Pat. No. 5,641,670; WO 96/29411; WO 94/12650; Koller, et al.,(1989); and Zijlstra, et al. (1989); U.S. Pat. Nos. 6,054,288;6,048,729; 6,048,724; 6,048,524; 5,994,127; 5,968,502; 5,965,125;5,869,239; 5,817,789; 5,783,385; 5,733,761; 5,641,670; 5,580,734; and inWO96/29411, WO 94/12650; and Koller, et al., (1994).

GENSET gene expression in mammalian cells, preferably human cells, maybe rendered defective, or alternatively may be altered by replacingendogenous GENSET polypeptide-encoding genes in the genome of an animalcell by a GENSET polypeptide-encoding polynucleotide according to theinvention. These genetic alterations may be generated by homologousrecombination using previously described specific polynucleotideconstructs.

Mammal zygotes, such as murine zygotes may be used as cell hosts. Forexample, murine zygotes may undergo microinjection with a purified DNAmolecule of interest. In addition, any one of the polynucleotides of theinvention may be introduced in an embryonic stem (ES) cell line,preferably a mouse ES cell line. ES cells, and methods of theirisolation and maintenance, are well known in the art, and are described,inter alia, in Abbondanzo et al., (1993), Meth. Enzymol., AcademicPress, New York, pp 803-823, Robertson, (1987), Embryo-derived stem celllines; In: E. J. Robertson Ed. Teratocarcinomas and embrionic stemcells: a practical approach. IRL Press, Oxford, pp. 71, and Pease andWilliam, (1990), Exp. Cell. Res. 190: 209-211.

Transgenic Animals

The terms “transgenic animals” or “host animals” are used herein todesignate animals that have their genome genetically and artificiallymanipulated so as to include one of the nucleic acids according to theinvention. The cells affected may be somatic, germ cells, or both.Preferred animals are non-human mammals and include those belonging to agenus selected from Mus (e.g. mice), Rattus (e.g. rats) and Oryctogalus(e.g. rabbits). In one embodiment, the invention encompasses non-humanhost mammals and animals comprising a recombinant vector of theinvention or a GENSET gene disrupted by homologous recombination with aknock out vector.

Thus, the present invention also concerns a transgenic animal containinga nucleic acid, a recombinant expression vector, or a recombinant hostcell according to the invention. A further object of the inventioncomprises recombinant host cells obtained from a transgenic animaldescribed herein.

Such transgenic animals may be good experimental models in order tostudy the diverse pathologies related to the increase or decrease of theexpression of a given GENSET gene. The transgenic animals may also beused to express a desired polypeptide of interest under the control ofthe regulatory polynucleotides of the GENSET gene, leading to highyields in the synthesis of this protein of interest, or totissue-specific expression of the gene.

In one embodiment, transgenic animals of the present invention areproduced by inserting a recombinant polynucleotide of the invention intoan embryonic or ES stem cell line (see, e.g., Thomas, et al. (1987) Cell51:503-512; Mansour et al., (1988) Nature 336:348-352), and isolating,cloning and injecting positive cells into blastocysts, which are theninserted into a female host animal and allowed to grow to term. For moredetails regarding the production of transgenic animals, and specificallytransgenic mice, see U.S. Pat. Nos. 4,873,191; 5,464,764; 5,789,215,Bradley (1987; In: E. J. Robertson (Ed.), Teratocarcinomas and embryonicstem cells: A practical approach. IRL Press, Oxford, pp. 113, Wood, etal. (1993), PNAS, 90: 4582-4585, Nagy et al., (1993), PNAS 90:8424-8428.

In another embodiment, transgenic animals are produced by microinjectingpolynucleotides into a fertilized oocyte. Methods for culturingfertilized oocytes to the pre-implantation stage are described, e.g., byGordon, et al. ((1984) Methods in Enzymology, 101, 414); Hogan, et al.((1986) in Manipulating the mouse embryo, A Laboratory Manual. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (for the mouseembryo)); Hammer, et al. ((1985) Nature, 315, 680 (for rabbit andporcine embryos)); Gandolfi, et al. ((1987) J. Reprod. Fert. 81, 23-28);Rexroad, et al. ((1988) J. Anim. Sci. 66, 947-953) (for ovine embryos));and Eyestone, et al. ((1989) J. Reprod. Fert. 85, 715-720); Camous etal. ((1984) J. Reprod. Fert. 72, 779-785); and Heyman, et al. ((1987)Theriogenology 27, 5968 (for bovine embryos)). Pre-implantation embryosare then transferred to an appropriate female by standard methods topermit the birth of a transgenic or chimeric animal, depending upon thestage of development when the transgene is introduced.

In a preferred embodiment of the present invention, transgenic mammalsare generated that secrete recombinant GENSET polypeptides in theirmilk. Preferably, expression in the mammary gland is accomplished byoperably linking the polynucleotide encoding the GENSET polypeptide to amammary gland specific promoter (e.g., from a casein or lactoglobulingene) and, optionally, other regulatory elements. Promoter and otherregulatory sequences, vectors, and other relevant teachings areprovided, e.g., by Clark (1998) J Mammary Gland Biol Neoplasia 3:337-50;Jost, et al. (1999) Nat. Biotechnol 17:160-4; U.S. Pat. Nos. 5,994,616;6,140,552; 6,013,857; Sohn, et al. (1999) DNA Cell Biol. 18:845-52; Kim,et al. (1999) J. Biochem. (Japan) 126:320-5; Soulier, et al. (1999)Euro. J. Biochem. 260:533-9; Zhang, et al. (1997) Chin. J. Biotech.13:271-6; Rijnkels, et al. (1998) Transgen. Res. 7:5-14; Korhonen, etal. (1997) Euro. J. Biochem. 245:482-9; Uusi-Oukari, et al. (1997)Transgen. Res. 6:75-84; Hitchin, et al. (1996) Prot. Expr. Purif.7:247-52; Platenburg, et al. (1994) Transgen. Res. 3:99-108; Heng-Cherl,et al. (1993) Animal Biotech. 4:89-107; and Christa, et al. (2000) Euro.J. Biochem. 267:1665-71.

In another embodiment, the polypeptides of the invention can be producedin milk by introducing polynucleotides encoding the polypeptides intosomatic cells of the mammary gland in vivo, e.g. mammary secretingepithelial cells. For example, plasmid DNA can be infused through thenipple canal, e.g. in association with DEAE-dextran (see, e.g., Hens, etal. (2000) Biochim. Biophys. Acta 1523:161-171), in association with aligand that can lead to receptor-mediated endocytosis of the construct(see, e.g., Sobolev, et al. (1998) 273:7928-33), or in a viral vectorsuch as a retroviral vector, e.g. the Gibbon ape leukemia virus (see,e.g., Archer, et al. (1994) PNAS 91:6840-6844). In any of theseembodiments, the polynucleotide may be operably linked to a mammarygland specific promoter, as described above, or, alternatively, anystrongly expressing promoter such as CMV or MoMLV LTR.

The polynucleotides used in such embodiments can either encode afull-length GENSET protein or a GENSET fragment. Typically, the encodedpolypeptide will include a signal sequence to ensure the secretion ofthe protein into the milk.

Uses of Polypeptides of the Invention

Protein of SEQ ID NO:2 (Internal Designation Clone 243525116-119-1-0-G6-F)

The cDNA of Clone 243525_(—)116-119-1-0-G6-F (SEQ ID NO:1) encodesprotein PCNAlt of SEQ ID NO:2, comprising the amino acid sequence:MPSGEFARICRDLSHIGDAVVISCAKDGVKFSA SGELGNGNIKLSQTSNVDKEEEAVTIEMNEPVQLTFALRYLNFFTKATPLSSTVTLSMSADVPLVVEYKIADMGHLKYYLAPKIEDEEGS.Accordingly, it will be appreciated that all characteristics and uses ofpolypeptides of SEQ ID NO:2 described throughout the present applicationalso pertain to the polypeptides encoded by the human cDNA included inClone 243525_(—)116-119-1-0-G6-F. In addition, it will be appreciatedthat all characteristics and uses of the polynucleotides of SEQ ID NO:1described throughout the present application also pertain to the nucleicacids comprising the human cDNA in Clone 243525_(—)116-119-1-0-G6-F. Apreferred embodiment of the invention is directed toward thecompositions of SEQ ID NO:1, SEQ ID NO:2, and Clone243525_(—)116-119-1-0-G6-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

The cDNA of SEQ ID NO: 1 is a splice variant of the human proliferatingcell nuclear antigen (PCNA), encoding a 123 amino-acid protein of SEQ IDNO:2 named PCNAlt. The protein of SEQ ID:2 comprises the PCNA C-terminaldomain.

Mammalian PCNA is so named because of its initial discovery as a cellcycle-dependent antigen (Miyachi et al, 1978). PCNA plays a criticalrole in several biological processes that appear disparate but have incommon a role in DNA metabolism. PCNAlt is a highly conserved essentialcomponent of the DNA replication machinery. The subcellular distributionof PCNAlt changes through the cell cycle, localizing to sites of DNAreplication in S-phase of cell cycle. PCNAlt is a nuclear proteinrequired for leading strand DNA synthesis by DNA polymerase delta, whichis part of the essential pathway for DNA replication. Consistent withits essential role in DNA replication, the expression of PCNAlt is verylow in quiescent and senescent cells but increases after stimulation byserum or by various growth factors. In addition, PCNAlt and polymerasedelta also play essential roles in the repair of damaged DNA. PCNAltlevels are elevated in response to DNA damage in vivo, the proteinrelocalizes to sites of DNA repair following damage. A direct role ofPCNAlt in nucleotide excision repair has been demonstrated.Co-ordination of these two roles of PCNAlt is thus essential for themaintenance of genetic integrity. In addition, PCNAlt can interact witha variety of cellular proteins involved in cell cycle regulation andcheck point control, such as cyclins and cyclin-dependent kinases.

In one embodiment, the protein of the invention or part thereof is usedto quantify in vitro the amount of cell proliferation in a biologicalsample. PCNAlt is thus used in assays and diagnostic kits for thequantification of cell proliferation in tissue samples such as a tissuesection or in cell cultures. The method comprises the steps ofcontacting a biological sample with a detectably labeled compoundcapable of selectively binding PCNAlt polypeptides or polynucleotides.For example, a polyclonal or monoclonal antibody, a PCNAlt-bindingfragment thereof, or a nucleic acid probe may be used. Preferably,antibody used in this embodiment is directed against nonlinear epitopesof PCNAlt. Preferably, the antibody used in this embodiment specificallybinds to PCNAlt polypeptides and not to PCNA polypeptides. Detection canbe carried out directly or indirectly with known immunohistological orimmunofluorescence techniques. Reagents contained in a kit according tothe invention can be directly labeled with generally known molecules,including, but not limited to, enzymes such as alkaline phosphatase andperoxidase and fluorescent dyes such as FITC, rhodamine, and Texas-Red.However, labeling can also occur indirectly by using primary antibodieslabeled with molecules such as biotin or digoxigenin which are thendetected with a secondary reagent.

In another embodiment, the current invention provides a method ofeffectively blocking proliferation or inhibiting the growth of a cell invivo or in vitro. Preferably, the present invention can be used to stopunrestrained cell proliferation and to eliminate as many tumor cells aspossible. More preferably, it is used to inhibit cell proliferationindependent of Gadd45 and p21 control. The method will be performed byadministering an antisense oligonucleotide directed against PCNAlt intothe cell. Such a method comprises the step of introducing apolynucleotide construct comprising an antisense oligonucleotidedirected against PCNAlt to a cell. One strategy for delivering antisenseoligonucleotides to targeted cells involves encapsulation orincorporation of the therapeutic bioactive molecules in liposomes, suchas cationic liposomes. These liposomes are known to provide a shieldagainst nucleotide degradation in vivo and can be targeted to specificareas of the body at which point they slowly release their contents.Alternatively, a polynucleotide construct comprising plasmid DNAoperably linked to the antisense oligonucleotide could be used.

The nucleotide sequences are administered in vivo in a suitable bufferor carrier solution known to those of skill in the art.

Any compound that inhibits or significantly decreases the expression ofPCNAlt polypeptides can also be used to inhibit replication within oneor more cells of the sample. Such compounds can be identified byscreening for test substances that decrease PCNAlt expression Thismethod of screening comprises the steps of contacting a cell with a testsubstance and comparing PCNAlt expression in the cell after exposure tothe test substance to that of an unexposed control cell. In addition,any compound that inhibits or significantly decreases the activity ofPCNAlt polypeptides can also be used to inhibit replication within oneor more cells of the sample. Such compounds can be identified byscreening for test substances that decrease PCNAlt activity comprisingthe steps of contacting a cell with a test substance and comparingPCNAlt activity on the cell replication after exposure to the testsubstance to that of an unexposed control cell. For example, inhibitorcompounds can represent molecules which disrupt the interaction betweenPCNAlt and their cellular targets involved in cell progression.

Alternatively, in another embodiment, the present invention provides amethod of effectively stimulating DNA replication in cells. The level oractivity of PCNAlt can be increased in cells to stimulate DNAreplication, thereby inducing cell proliferation. Preferably, PCNAlt isused to increase cell proliferation independent of Gadd45 and p21control. PCNAlt levels may be increased by introducing PCNAltpolynucleotides or polypeptides into a cell in an amount sufficient tospecifically stimulate DNA replication of one or more cells within thesample. Preferably, such methods can be performed in vitro to increaseproliferation of cells in culture. For example, it can be used tomaintain cell proliferation under serum starvation conditions that isrequired for some in vitro experiments. On the other hand, an increasedlevel of PCNAlt is also used to decrease cell proliferation specificallydependent on Gadd45 or p21 control. Whatever the case, the level ofPCNAlt can be increased in cells in any of a number of ways. Forinstance, purified PCNAlt protein may be introduced to the cells bymicroinjection or by liposome or micelle-mediated transport. Suchliposomal or micellar microcapsule may optionally be combined with acell type-specific target, such as an antibody or receptor ligand.Alternatively, PCNAlt polynucleotides may be introduced to a cell bymethods common to the art such as transfection, electroporation, orviral transduction. The vectors mentioned above may also be used tointroduce PCNAlt polynucleotides to a cell.

The present invention also provides animal models generated bymodulating the expression or activity of the present protein in one ormore tissues of the animal. Preferably, PCNAlt expression can beselectively activated or inactivated in a particular cell type using aconditional expression system. These animals can be generated with anyinducible method of targeting inactivation of PCNAlt. Such animals areuseful for a number of purposes, because they represent an in vivo assaymethod for testing the efficacy of PCNAlt inactivation as a candidatestrategy potentially useful for the treatment of variouspathophysiological aspects of diseases specifically related to an excessof cell proliferation including the cancers listed herein. Such modelsare also extremely useful in the assessment of combinatory therapies,i.e. PCNAlt inactivation in addition to one or other compounds ofinterest. Malignancy conditions to be tested can be induced by anymethod well known in the art such as chemicals, expression of variousoncogenes by transgenesis for example, genotoxic agents (UV, gammairradiation), etc.

In another embodiment, the current invention is used to diagnosediseases or disorders associated with unrestrained cell proliferationand in particular in various cancers. Aberrant PCNAlt expression orlocalization is a significant prognostic indicator, and is preferablyused as a prognostic factor for the clinical course of various cancers.More preferably, it can be measured in case of tumors linked to p53inactivation. In one such embodiment, the present invention can alsoprovide indicators that are suitable to estimate the efficacy orresponsiveness to a form of chemotherapy. The method of detection maypreferably occur directly on a section of the sample. The samplematerial can be present for example as a biopsy, a tissue homogenate, afine-needle aspirate or tumor resection or can be performed by flowcytometry. The method comprises the steps of contacting a tissue sampleobtained from an individual suspected of suffering from the disease orcondition or at risk of developing the disease or condition, with adetectably labeled compound capable of selectively binding PCNAltpolypeptides or nucleic acids. For example, a polyclonal or monoclonalantibody or PCNAlt-binding fragment thereof or a nucleic acid probe maybe used. Preferably, the antibodies used in one such embodiment aredirected against nonlinear epitopes of PCNAlt. Preferably, theantibodies specifically bind to PCNAlt polypeptides and not to PCNApolypeptides. Detection can be carried out directly or indirectly withknown immunohistological and immunofluorescent techniques. Reagentscontained in a kit according to the invention can be directly labeledwith generally known molecules, including, but not limited to enzymessuch as alkaline phosphatase and peroxidase and fluorescent dyes such asFITC, rhodamine, and Texas-Red. However, labeling can also occurindirectly by using secondary antibodies labeled with molecules such asbiotin, digoxigenin or the like and are then detected with a secondaryreagent. PCNAlt immunoreactivity can be regarded as indicative ofneoplasia but preferably with at least one other marker. The secondmarker index to be detected is selected from the group comprising amarker for transformed cells, protein being overexpressed in aneoplastic cell, or a protein expressed in a form normally not presentin the cell. For example, the procedure can be adapted for doubleimmunostaining of PCNAlt together with alpha-actin, bromodeoxyuridine,keratin, type IV collagen and vimentin. In addition, the proteinrepresenting the second marker may preferably represent a tissuespecific marker. Thus, a kit may contain an antibody directed againstPCNAlt and antibodies against one or more of the markers mentionedabove. The condition of an individual can be monitored continuously andthe quantified amount of this particular protein measured in thepathological sample can be compared with the amount quantified in abiological sample of a normal individual or with previous samples fromthe same individual. The determination of all indices mentioned abovecan occur at the same time, sequentially or after one another.

In one other embodiment, the present invention is useful in diagnosingpatients with an excess amount of PCNAlt and monitor PCNAlt expressionin such conditions. Preferably, the present invention provides a methodto diagnose and the monitor diseases such as systemic lupuserythematosis disease and malignant lymphomas. The method comprises thesteps of contacting a tissue sample obtained from an individualsuspected of suffering from the disease or condition or at risk ofdeveloping such a disease or condition, with a detectably labeledcompound capable of selectively binding PCNAlt polypeptides or nucleicacids. For example, a polyclonal or monoclonal antibody or aPCNAlt-binding fragment thereof or a nucleic acid probe may be used.Preferably, the antibodies used in one such embodiment are directedagainst nonlinear epitopes of PCNAlt. Preferably, the antibodiesspecifically bind to PCNAlt polypeptides and not to PCNA polypeptides.Thus, the amount of PCNAlt can be monitored and quantified in a subjectin the pathological sample and be compared with the amount quantified ina biological sample of a normal individual.

A further embodiment of the present invention is to provide novelmethods and compositions useful for the treatment or prevention ofdiseases and conditions related to excessive cell proliferation andpreferably with malignancy conditions. Such methods comprise theadministration of a therapeutically-effective amount of antisense PCNAltoligonucleotides to mammals suffering from the disease or condition,where “effective amount” is meant a concentration of antisenseoligonucleotides capable of significantly reducing cell proliferation.The compositions of the invention are preferably delivered to anindividual in a pharmaceutically acceptable carrier, such as a salinesolution or other physiological buffer suitable for administration to apatient. The particular amount of the compositions of the invention thatwill be administered to the mammal for any particular condition willdepend on the clinical condition of the patient, and other factors suchas the weight, age, and route of delivery. Such compositions can beadministered by any suitable route. For treatment purposes, thecomposition can also be incorporate into liposomes, such as cationicliposomes to deliver the therapeutic bioactive molecules to targetedcells. These compositions can comprise antisense oligonucleotidesdirected against PCNAlt, and, optionally, one or more other compounds ofinterest. This co-administration may be by simultaneous administrationor by separate or sequential administrations. All of these additionalcomponents may be either obtained from natural sources or produced byrecombinant genetic engineering techniques and/or chemical modification.

Protein of SEQ ID NO:4 (Internal Designation Clone 643144181-17-2-0-A12-F)

The cDNA of Clone 643144_(—)181-17-2-0-A12-F (SEQ ID NO:3) encodesLectir of SEQ ID NO:4, comprising the amino acid sequence:MQDEDGYITLNIKTRKPALVSVGSASSSWWRVMALILLILCVGMVVGLVALGIWSVMQRNYLQDENENRTGTLQQLAKRFCQYVVKQSELKGTFKGHKCSPCDTNRWRYYGDSCYGFFRHNLTWEESKQYCTDMNATLLKIDNRNIVEYIKARTHLIRWVGLSRQKSNEVWKWEDGSVISENMFEFLEDGKGNMNCAYFHNGKMHPTFCENKHYLMCERKAGMTKVDQLP. Accordingly, it willbe appreciated that all characteristics and uses of the polypeptides ofSEQ ID NO:4 described throughout the present application also pertain tothe polypeptides encoded by the human cDNA included in Clone643144_(—)181-17-2-0-A12-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:3 describedthroughout the present application also pertain to the nucleic acidscomprising the human cDNA in Clone 643144_(—)181-17-2-0-A 12-F. Apreferred embodiment of the invention is directed toward thecompositions of SEQ ID NO:4, SEQ ID NO:3, and Clone643144_(—)181-17-2-0-A12-F.

Lectir is a polymorphic variant of the C-type Lectin-like receptor 2(GenBank accession number AAF36777). Lectir possesses an anchor signal,a transmembrane domain (VMALILLILCVGMVVGLVALGIW) (SEQ ID NO:53), alectin C-type domain(QYCTDMNATLLKIDNRNIVEYIKARTHLIRWVGLSRQKSNEVWKWEDGSVISENM FEFLEDGKGNMNCAYFHNGKMHPTFCENKHYLMCE) (SEQ ID NO:54) and an extracellularlink domain (RHNLTWEESKQYCTDMNATL) (SEQ ID NO:55). Lectir is homologousto the oxidized LDL receptor (LOX-1). Lectir is expressed in myeloidcells such as monocytes, dendritic cells and granulocytes.

Lectir is involved in antigen capture and phagocytosis of apoptoticbodies. Phagocytosis of aged and apoptotic cells is an essential processto protect normal healthy cells from the harmful contents and debris ofdying cells. Apoptosis is accompanied by the clustering of intracellularautoantigens, which are selectively-cleaved and phosphorylated, and bythe exposure of anionic phospholipids such as phosphatidyl-serine.Modified lipoproteins (e.g., oxidized lipoproteins) play a crucial rolein apoptosis signaling. Lectins and C-type lectin like receptors areexpressed both by cells undergoing apoptosis and by phagocytic cells.Lectir is a phagocytic receptor that binds to phospholipids, capturesapoptotic bodies and directs them from the extracellular space to aspecialized processing compartment of myeloid cells.

An embodiment of the invention is directed to compositions comprising anantibody directed against a Lectir polypeptide or against a Lectirpolypeptide fragment. Preferably, the antibody specifically binds to theLectir polypeptide and not to the C-type Lectin-like receptor-2polypeptide. An additional preferred embodiment of the invention is amethod of binding Leclir polypeptides with a Leclir-specific antibody orLeclir-binding fragment thereof. This method comprises the step ofcontacting a Leclir polypeptide with a Leclir-binding antibody orLeclir-binding fragment thereof under conditions that allow binding.This method may be applied to detection and purification of Leclir, aswell as targeting Leclir-expressing cells. These aspects are discussedin detail herein.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a Leclir polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing Leclir expression.Preferably, the polynucleotides capable of directing Leclir expressionare located in the 5′ regulatory region of the Leclir gene. Furtherpreferably, these polynucleotides are located within 500 base pairs ofthe Leclir coding region. These polynucleotides preferably comprise apromoter sequence.

An embodiment of the present invention includes methods of purifyingLectir polypeptides. This method comprises the steps of: i) obtaining acell capable of expressing a Lectir polypeptide; ii) growing said cellunder conditions suitable to produce said polypeptide; and iii)purifying said polypeptide. An example of this method comprises thesteps of: i) transfecting a mammalian host cell with a recombinantexpression vector comprising a polynucleotide of the present invention,and ii) purifying the produced protein. Leclir purification of theprotein can be done following any technique well-known to those skilledin the art. Preferably, a Leclir-binding antibody or an antigen bindingfragment thereof may be bound to a chromatographic support to form anaffinity chromatography column. Preferably, the antibodies are thosedescribed above.

An embodiment of the present invention relates to methods of using thepolypeptides and the polynucleotides of the present invention to enhancephagocytosis of accumulating apoptotic cells. Any compositions andmethods containing, e.g., Lectir polypeptide or part thereof, apolynucleotide encoding the protein, or a compound that increases theexpression or activity of Lectir can be used.

In a preferred embodiment, a Lectir polypeptide is used in a method toenhance phagocytosis of apoptotic cells. This method comprises the stepof introducing Lectir polypeptide to a cell. Preferred cells arephagocytic cells, such as dendritic cells and macrophages. Lectirpolypeptides may be introduced by introducing a polynucleotide constructcomprising polynucleotides that encode Lectir polypeptides or abiologically active fragment thereof to a cell. This method may bedirected to the removal of apoptotic cells from a sample of cells to becultured. Additionally, this method may be applied to reduce theseverity of diseases that result from cellular accumulation such asrheumatoid arthritis, systemic lupus erythematosus, psoriatic arthritis,asthma, bronchitis, sarcoidosis, and pulmonary fibrosis. This method mayalso be applied when treating a patient with apoptosis-inducing drugssuch as, e.g., cisplatin and carboplatin, for reducing cellularaccumulation of apoptotic cells. Notably, apoptosis-inducing drugs arewidely used for treating tumor proliferation.

In another embodiment, the methods of the present invention relate tothe administration of a recombinant expression vector comprising one ofthe polynucleotides of the invention to a patient suffering from adisease associated with accumulation of apoptotic cells. Preferredexpression vectors include viral vectors, especially adenoviral andlentiviral vectors.

In still another embodiment, genetic modification of a cell with avector comprising one of the polynucleotides of the invention may beaccomplished using one or more techniques well known in the gene therapyfield. For example, one of the methods described in Mulligan (Mulligan,Science, 260:926-32 (1993)), which disclosure is hereby incorporated byreference in its entirety, can be used.

In still another embodiment, the compositions of the present inventioncomprise a substance that increases Lectir expression. Additionally, themethods of the present invention relate to methods of screening testsubstances that increase Lectir expression. These methods comprise thesteps of: i) contacting a cell with a test substance; and ii) comparingLectir expression in the cell after exposure to the test substance tothat of an unexposed control cell. Preferably, the test substancemodifies the expression of Lectir in monocytes, dendritic cells and/orgranulocytes while not in other cell types.

Another embodiment of the present invention is directed to compositionscomprising substances that increase Lectir phagocytic activity.Additionally, the methods of the present invention relate to methods ofscreening test substances that increase Lectir expression. These methodscomprise the steps of: i) contacting a cell with a test substance; andii) comparing Lectir activity in the cell after exposure to the testsubstance to that of an unexposed control cell. Preferably, the testsubstance modifies Lectir activity in monocytes, dendritic cells and/orgranulocytes while not in other cell types. Several methods, includingbut not limited to the method described by Simpson et al. (J ImmunolMethods 29:221-6 (1979)), which disclosure is hereby incorporated byreference in its entirety, are available for quantifying phagocyticactivity. Such a method can also be used for verifying in vitroeffectiveness of compositions and methods of the present invention toenhance phagocytosis.

In a preferred embodiment, Lectir polypeptides or a substance thatincreases Lectir expression or Lectir activity can be processed inaccordance with conventional methods of pharmacy to produce medicinalagents for administration to patients. Thus, the pharmaceuticalcomposition comprising Lectir or part thereof or a substance thatincreases Lectir expression may be made up in a solid form (e.g.granules for oral administration, powders for inhalation) or in a liquidform (e.g. solutions for oral administration or for injection).Effective doses of the compositions of the present invention fortreating a patient suffering from disorders associated with accumulationof apoptotic cells can be determined according to the relevanttechniques.

Another embodiment of the present invention relates to methods of usingan antibody directed against Lectir polypeptides for purifying myeloidcells. Preferred antibodies are those described above. Such a method formyeloid cells comprises the steps of: i) labeling an Lectir-specificantibody with a molecule that can be used to provide a detectablesignal, ii) running a body fluid through a sorting apparatus (e.g., afluorescence-activated cell sorter or a magnetic activated cell-sortingapparatus, containing the labeled anti-Lectir antibody). Myeloid cells,which include monocytes, granulocytes, and megakaryocytes monitor forthe presence of foreign bodies, provide protection against neoplasticcells, scavenge foreign materials, and produce platelets. A highlypurified population of myeloid cells is necessary for a variety of invitro experiments and in vivo indications. For example, myeloid cellsfind use in i) enriching the hematopoietic system of a host deficient inany class of myeloid cells; and ii) detecting diseases associated withmyeloid cell dysfunction.

Protein of SEQ ID NO:6 (Internal Designation Clone 212950.cREC116-075-2-0-H1-F)

The cDNA of Clone 212950.cREC_(—)116-075-2-0-H1-F (SEQ ID NO:5) encodesvlADAM20 of SEQ ID NO:6, comprising the amino acid sequence:MVQLHQDTDPQIPKGQPCTLNSSEGGARPAVPHTLFSSALDRWLHNDSFIMAVGEPLVHIRVTLLLLWFGMFLSISGHSQARPSQYFTSPEVVIPLKVISRGRGAKAPGWLSYSLRFGGQRYIVHMRVNKLLFAAHLPVFTYTEQHALLQDQPFIQDDCYYHGYVEGVPESLVALSTCSGGFLGMLQINDLVYEIKPISVSATFEHLVYKIDSDDTQFPPMRCGLTEEKIAHQMELQLSYNFTLKQSSFVGWWTHQRFVELVVVVDNIRYLFSQSNATTVQHEVFNVVNIVDSFYHPLEVDVILTGIDIWTASNPLPTSGDLDNVLEDFSIWKNYNLNNRLQHDVAHLFIKDTQGMKLGVAYVKGICQNPFNTGVDVFEDNRLVVFAITLGHELGHNLGMQHDTQWCVCELQWCIMHAYRKVTTKFSNCSYAQYWDSTISSGLCIQPPPYPGNIFRLKYCGNLVVEEGEECDCGTIRQCAKDPCCLLNCTLHPGAACAFGICCKDCKFLPSGTLCRQQVGECDLPEWCNGTSHQCPDDVYVQDGISCNVNAFCYEKTCNNHDIQCKEIFGQDARSASQSCYQEINTQGNRFGHCGIVGTTYVKCWTPDIMCGRVQCENVGVIPNLIEHSTVQQFHLNDTTCWGTDYHLGMAIPDIGEVKDGTVCGPEKICIRKKCASMVHLSQACQPKTCNMRGICNNKQHCHCNHEWAPPYCKDKGYGGSADSGPPPKNNMEGLNVMGKLRYLSLLCLLPLVAFLLFCLHVLFKKRTKSKEDEEG. Accordingly, it will beappreciated that all characteristics and uses of the polypeptides of SEQID NO:6 described throughout the present application also pertain to thepolypeptides encoded by the human cDNA included in Clone212950.cREC_(—)116-075-2-0-H1-F. In addition, it will be appreciatedthat all characteristics and uses of the polynucleotides of SEQ ID NO:5described throughout the present application also pertain to the nucleicacids comprising the human cDNA in Clone212950.cREC_(—)116-075-2-0-H1-F. A preferred embodiment of the inventionis directed toward the compositions of SEQ ID NO:6, SEQ ID NO:5, andClone 212950.cREC_(—)116-075-2-0-H1-F.

Preferred vlADAM20 polypeptides for uses in the methods described belowinclude the polypeptides comprising the amino sequence of:MVQLHQDTDPQIPKGQPCTLNSSEGGARPAVPHTLFSSALDRWLHNDSFI (SEQ ID NO:56).

Also preferred are the polypeptide fragments having a biologicalactivity of proteolytically processing sperm surface proteins and thepolynucleotides encoding the fragments.

vlADAM20 is a sperm protein that is a member of the ADAM family, andthat displays all structural elements that are characteristic of thisfamily. Notably, vlADAM20 displays a catalytic site of an activeZn²⁺-metalloprotease (HELGHNLGMQHD) (SEQ ID NO:57), one disintegrinconsensus sequence (EEGEECDCG) (SEQ ID NO:58) and one transmembranedomain. The ADAM family of proteins contains a disintegrin andmetalloproteinase domain. Members of this family are cell surfaceproteins with a unique structure possessing both potential adhesion andprotease domains. Although all ADAMs have the same domain organization,members of the ADAM do not all display the same functions. The functionsdisplayed by members of the ADAM family are regulated by processes thatinclude alternative splicing, differential gene expression,dimerization, and proteolytic processing. ADAMs are involved in diversebiological processes such as spermatogenesis, fertilization, myoblastfusion and neuron proliferation (Wolfsberg et al, Dev Biol. 180:389-401(1996)).

vlADAM20 is a novel membrane-anchored sperm surface protease that isinvolved in spermatogenesis. vlADAM20 is involved in remodeling thedeveloping sperm surface, i.e., proteolytically processing other spermsurface proteins.

An embodiment of the invention is directed to a composition comprising avlADAM20 polypeptide sequence of SEQ ID NO:6.

A further embodiment of the invention is directed to a compositioncomprising a vlADAM20 polypeptide fragment having biological activity ofproteolytically processing sperm surface proteins.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:5 encoding a vlADAM20polypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a vlADAM20 polypeptidefragment having biological activity of proteolytically processing spermsurface proteins.

A further embodiment of the invention is directed to an antibody or anantigen-binding fragment that binds to a vlADAM20 polypeptide or againsta vlADAM20 polypeptide fragment. Preferably, the antibody specificallybinds to the vlADAM20 polypeptide and not to the ADAM20 polypeptide.Preferably, the antibody or antigen-binding fragment recognizes anepitope comprising one or more of the 50 amino-acids located at theamino-terminal extremity of vlADAM20, wherein one or more of theseamino-acids are required for antibody binding. Even more preferably, theantibody recognizes the QLHQDTDPQIPKGQPCT (SEQ ID NO:59) amino-acidsequence or the LNSSEGGAR (SEQ ID NO:60) amino-acid sequence. As usedherein, the term anti-vlADAM20 antibody includes both intact moleculesas well as active fragments thereof, such as those capable of bindingantigens.

The present invention also relates to a method of binding ananti-vlADAM20 antibody to a vlADAM20 polypeptide comprising the step of:contacting a vlADAM20 polypeptide with said antibody or antigen-bindingfragment thereof under conditions that allow binding to take place. Suchconditions are well known to those skilled in the art. Additionally, thepresent invention also relates to a method of binding an anti-vlADAM20antibody or antigen-binding fragment thereof to a cell expressing avlADAM20 polypeptide or fragment thereof comprising the step of:contacting a cell expressing a vlADAM20 polypeptide or part thereof withsaid antibody or antigen-binding fragment under conditions that allowbinding to take place. Preferably, the antibody or fragment used inthese methods is vlADAM20-specific, as discussed above. Such methods arefor example useful for purifying testis cells as further describedherein.

Another embodiment of the present invention relates to methods of usingcompositions comprising an antibody directed against vlADAM20polypeptides for purifying testis cells. Preferably, such compositionscomprise the preferred antibodies described above. Such a method forpurifying testis cells comprises the steps of: i) labeling by standardmethods of the anti-vlADAM20 antibody with a molecule that can be usedto provide a detectable signal, ii) running a biopsied testis sample ormammalian cells through a sorting apparatus, e.g. afluorescence-activated cell sorter or a magnetic activated cell-sortingapparatus, containing the labeled anti-vlADAM20 antibody. Purifyingtestis cells are useful for in vitro analysis and diagnosis of variousdiseases associated with testis including, but not limited to,testicular cancer, testicular intraepithelial neoplasia, testicularmicrolithiasis and sterility.

An embodiment of the present invention relates to a method of producingvlADAM20 polypeptides comprising the steps of: i) transfecting a hostcell with a recombinant expression vector comprising a polynucleotide ofthe present invention; and optionally ii) purifying the producedprotein. The purification of the protein can be done following anytechnique well known to those skilled in the art. Preferably, anantibody directed against vlADAM20 or part thereof may be bound to achromatographic support to form an affinity chromatography column. Evenmore preferably, the antibody recognizes the 50 amino-terminal aminoacids of vlADAM20. Alternatively, vlADAM20 polypeptides may be producedby a method comprising the step of: i) transfecting a host cell with apolynucleotide capable of directing vlADAM20 expression. Preferably, thepolynucleotides capable of directing vlADAM20 expression are located inthe 5′ regulatory region of the vlADAM20 gene. Further preferably, thesepolynucleotides are located within 500 base pairs of the vlADAM20 codingregion. These polynucleotides preferably comprise a promoter sequence.Techniques known in the art for introducing polynucleotide sequences toendogenous sequences are described in U.S. Pat. No. 5,641,670 and PCTWO9629411, which disclosures are hereby incorporated by reference intheir entireties.

In an embodiment, a vlADAM20 polypeptide or a biologically activefragment thereof can be used in a “cocktail” of proteases that is ableto digest a wide range of proteins. Such protease cocktails are usefulin laboratory assays to degrade undesirable proteins in a sample, forexample for removing proteins in a DNA preparation or for removingenzymes after any enzymatic reaction.

In another embodiment, vlADAM20 or a biologically active fragmentthereof can be used in combination with a detergent for the removal ofstains having a protein component, similar to the use of proteasesdescribed in U.S. Pat. No. 5,599,400, which disclosure is incorporatedby reference in its entirety. The composition can contain knowndetergent constituents such as, e.g. surfactants, foam enhancers andfillers. The detergent preferably contains between 0.001% to 10%vlADAM20 polypeptides. vlADAM20 polypeptides can be included in adetergent composition or can be combined with other constituents at thetime of use as an additive. The detergent additive can be formulated asa liquid, powder, granulate or slurry.

Still another embodiment relates to compositions and methods for usingvlADAM20 polypeptides as a contraceptive immunogen. It was shown thatmen and women who spontaneously produce anti-sperm antibodies areinfertile but otherwise healthy (Bronson et al, Fertil Steril. 42:171-83(1984)). Such a method comprises the step of: administering to anindividual a vlADAM20 polypeptide as described in U.S. Pat. No.6,197,940, which disclosure is hereby incorporated in its entirety, inan effective amount to reduce the fertility of that individual viageneration of antibodies to vlADAM20. Preferably, immunogenic vlADAM20polypeptides are used in such compositions. The administered amount ofimmunogenic vlADAM20 polypeptides depends upon factors such as route ofadministration, species, and the use of booster administration. Forexample, a dose of about 0.1 to 100 micrograms of vlADAM20 polypeptidesper kg of body weight may be used. Compositions of the present inventionmay be prepared as both human and veterinary vaccine formulations.Contraceptive vaccines can be produced by combining vlADAM20polypeptides with a pharmaceutically suitable carrier and with anadjuvant that contains non-specific stimulators of the immune systemsuch as, e.g., immunogenic fragments of Bordatella pertussis.

Compositions of the present invention may be made up in a solid form(e.g. granules for oral administration) or in a liquid form (e.g.solutions for oral administration or for injection). Compositions of thepresent invention are included in the carrier in an amount that iseffective to reduce the fertility of the subject being treated.Compositions of the present invention may also comprise othercontraceptive molecules. For example, a female contraceptive vaccinecomprising vlADAM20 polypeptides may also comprise PH30 beta chainproteins that are described in U.S. Pat. No. 5,693,496, which disclosureis incorporated hereby by reference in its entirety, and a malecontraceptive vaccine may also comprise SP-22 polypeptides that aredescribed in U.S. Pat. No. 6,197,940, which disclosure is incorporatedhereby by reference in its entirety.

A further embodiment of the present invention is directed to substancesthat decrease vlADAM20 expression, and to a method of screening for suchsubstances comprising the steps of: i) contacting a cell with a testsubstance, ii) comparing vlADAM20 expression in an exposed cell to thatof an unexposed control cell, iii) quantifying said expression levels,and iv) determining the ratio of vlADAM20 expression in an exposed cellrelative to the expression in an unexposed cell. Preferably, vlADAM20expression is studied in testis cells. Preferably, compositionscomprising substances that decrease vlADAM20 expression can beadministered to male individuals for contraceptive purposes.

A further embodiment of the present invention is directed to substancesthat decrease vlADAM20 activity, and to a method of screening for suchsubstances comprising the steps of: i) contacting a cell with a testsubstance, ii) comparing vlADAM20 proteolytic activity in an exposedcell to that of an unexposed control cell, iii) quantifying saidexpression levels, and iv) determining the ratio of vlADAM20 proteolyticactivity in an exposed cell relative to the expression in an unexposedcell. Preferably, vlADAM20 expression is studied in testis cells.vlADAM20 proteolytic activity can for example be measured by the alpha2M complex formation assay as described by Frosty Loechel et al (J BiolChem 273:16993-7 (1998)). Preferably, compositions comprising substancesthat decrease vlADAM20 expression can be administered to maleindividuals for contraceptive purposes.

As used herein, the term antagonist includes substances that thatdecrease vlADAM20 expression, substances that decrease vlADAM20activity, and antibodies or antigen-binding fragments that inhibit theactivity of vlADAM20.

Another embodiment of the present invention relates to methods of usingcompositions comprising a vlADAM20 antagonist for blocking vlADAM20proteolytic activity. Preferably, the antagonist is one of the preferredantibodies described above. Such compositions can be administered to acell, a tissue sample or an individual. Preferably, such compositionsare administered to male individuals for blocking spermatogenesis incontraceptive purposes.

Still another embodiment of the present invention relates to methods ofusing compositions comprising an anti-vlADAM20 antibody foragglutinating and immobilizing sperm. Preferably, such compositionscomprise the preferred antibodies described above. Such compositions canbe administered to, e.g., a cell, a tissue sample or a patient.Preferably, such compositions are administered to female individuals forblocking fertilization in contraceptive purposes.

Protein of SEQ ID NO:8 (Internal Designation Clone 1000849866181-44-3-0-A9-F)

The cDNA of SEQ ID NO:7 of clone 1000849866_(—)181-44-3-0-A9-F encodesLipoglobulin of SEQ ID NO:8, comprising the sequence:MRSLGALLLLLSACLAVSAGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHKSKWNITMESYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRAPQLRETLLQDFRVVAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPRVRRAATPRRGRIRGWATGN. Accordingly, it will beappreciated that all characteristics and uses of the polypeptide of SEQID NO:8 described throughout the present application also pertains tothe polypeptide encoded by the human cDNA included in clone1000849866_(—)181-44-3-0-A9-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:7described throughout the present application also pertains to thepolypeptide encoded by the nucleic acids comprising the human cDNA inclone 1000849866_(—)181-44-3-0-A9-F. A preferred embodiment also pertaintoward the composition of SEQ ID NO:7, SEQ ID NO:8 and clone1000849866_(—)181-44-3-0-A9-F. Also preferred are polypeptides fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

The protein of SEQ ID NO:8 is a splice variant of the sequence of Alpha1 Microglobulin (swissprot accession number P02760). The 221 amino acidlipoglobulin protein displays a lipocalin motif and belongs to thelipocalin superfamily. These extra-cellular proteins bind and transportsmall hydrophobic ligands such as steroids, bilins, retinoids, andlipids. Lipocalins function in a variety of processes including nutrienttransport, cell growth regulation, immune response and prostaglandinsynthesis.

Lipoglobulin is a plasma glycoprotein. The protein carries a set ofchromophores which gives the protein a yellow-brown color and anextremely heterogeneous charge. Lipoglobulin can associate covalentlywith many plasma proteins like factor IX Zutphen, Factor XII Tenri andseveral protein C mutants. Lipoglobulin is translated in the liver celland secreted to the blood. Lipoglobulin is found in many physiologicalfluids including plasma, urine, and cerebrospinal fluid. The proteinappears not only as a free monomer but also in complexes with IgA andalbumin. Lipoglobulin is also involved in anti-inflammatory andimmunosuppressive activities. Lipoglobulin is one of the positiveacute-phase proteins, in that circulating levels of lipoglobulinincrease in response to stress and inflammatory stimulation.Lipoglobulin accumulates at sites of inflammation where it inhibitsplatelet and neutrophil activation and inhibits phagocytosis. Theimmunomodulatory properties of lipoglobulin are due to glycosylation.Lipoglobulin is 40% carbohydrate, making it unusually acidic andsoluble. The glycosylation pattern of lipoglobulin changes duringacute-phase response, and deglycosylated lipoglobulin has noimmunosuppressive activity. Lipocalins are used as diagnostic andprognostic markers in a variety of disease states. The plasma level ofLipoglobulin can be monitored during pregnancy and in diagnosis andprognosis of conditions including cancer chemotherapy, renaldysfunction, myocardial infarction, arthritis, and multiple sclerosis.

An additional preferred embodiment of the invention is a method ofbinding lipoglobulin polypeptides with a lipoglobulin-specific antibodyor lipoglobulin-binding fragment thereof. This method comprises the stepof contacting a lipoglobulin polypeptide with a lipoglobulin-bindingantibody or lipoglobulin-binding fragment thereof under conditions thatallow binding. This method may be applied to detection and purificationof lipoglobulin. These aspects are discussed in detail herein.

An embodiment of the present invention relates to compositionscomprising Lipoglobulin polypeptides. The method of producingLipoglobulin polypeptides comprises the steps of: i) transfecting a hostcell with a recombinant expression vector comprising a polynucleotide ofthe present invention, and ii) purifying the produced protein. Thepurification of the protein can be done following any technique wellknown to those skilled in the art. Preferably, an antibody directedagainst Lipoglobulin or part thereof, preferably, an antibody directedagainst the C-terminal sequence of Lipoglobulin polypeptide, may bebound to a chromatographic support to form an affinity chromatographycolumn.

In another embodiment, the invention provides methods and compositionsfor detecting the level of Lipoglobulin mRNA expression. Quantificationof mRNA levels of Lipoglobulin may be useful for the diagnosis orprognosis of diseases associated with an altered expression of theprotein of the invention. Conditions, diseases or disorders associatedwith altered expression include, but are not limited to, certaincancers, cystic fibrosis, ulcer, clotting disorders such as hemophelia,inflammation and immune based disorders, acquired immunodeficiencysyndrome (AIDS), autoimmune disorders such as arthritis, fertilitydisorders, and hypothyroidism.

Assays for the detection and quantification of the mRNA of the proteinof the invention are well known in the art (see, for example, Maniatis,Fitsch and Sambrook, Molecular Cloning; A Laboratory Manual (1982), orCurrent Protocols in Molecular Biology, Ausubel, F. M. et al. (Eds),Wiley & Sons, Inc.). For example, the nucleic acid molecule or probe maybe labeled by standard methods and added to a biological sample from apatient under conditions of formation of hybridization complexes. Afteran incubation period, the sample is washed and the amount of label (orsignal) associated with hybridization is quantified and compared with astandard value. If the amount of label in the patient sample issignificantly altered in comparison to the standard value, then thepresence of the associated condition, disease or disorder is indicated.Such assays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies and in clinical trialand to monitor the treatment of an individual patient. Once the presenceof a condition is established and a treatment protocol is initiated,diagnostics assays may be repeated on a regular basis to determine ifthe level of expression in the patient begins to approximate that whichis observed in a normal subject. The results obtained from successiveassays may be used to show the efficacy of treatment over a periodranging from several days to months.

In another embodiment, the Lipoglobulin polypeptide or fragment thereofmay be used to diagnose, disorders characterized by the presence ofsubstrates, for example, Factor IX Zutphen, Factor XII Tenri and severalprotein C mutants. Such disorders include but are not limited to, factorXII deficiency and haemophilia B. In such a method, the Lipoglobulinpolypeptide or fragment thereof is used in assays and diagnostic kitsfor the identification and/or quantification of substrates such asFactor IX Zutphen, Factor XII Tenri, and protein C mutants in abiological sample.

In another embodiment, the invention relates to methods and compositionsfor detecting and quantifying the level of the protein of the inventionpresent in a particular biological sample. These methods are useful forthe diagnosis or prognosis of diseases associated with altered levels ofthe protein of the invention. Diagnostic assays to detect the protein ofthe invention may comprise a biopsy, in situ assay of cells from organor tissue sections, or an aspirate of cells from a tumor or normaltissue. In addition, assays may be conducted upon cellular extracts fromorgans, tissues, cells, urine, or serum or blood or any other body fluidor extract.

Detection and quantification of Lipoglobulin polypeptides using eitherspecific polyclonal or monoclonal antibodies are known in the art.Examples of such techniques include enzyme-linked immunoabsorbent assays(ELISAs), radioimmunoassays (RIAs), and fluorescence activated cellsorting (FACS). Example of an antibody is described in U.S. Pat. No.6,153,192, which disclosure is incorporated by reference in itsentirety.

In one embodiment, Lipoglobulin or a preferred fragment may beadministered to a subject to treat or prevent a condition associatedwith altered expression or activity of the protein of the invention.Examples of such conditions include, but are not limited to, thosedescribed above.

In another embodiment, a pharmaceutical composition comprising asubstantially purified Lipoglobulin polypeptide in conjunction with apharmaceutical carrier may be administered to a subject to treat orprevent a condition associated with altered expression or activity ofthe endogenous protein including, but not limited to, those providedabove.

In another embodiment, a substance which modulates the activity ofLipoglobulin may be administered to a subject to treat or prevent acondition associated with altered half life, expression, or activity ofLipoglobulin polypeptides including, but not limited to, those listedabove. In one aspect, an antibody which specifically binds Lipoglobulinmay be used as a targeting and delivery mechanism for bringing apharmaceutical agent to cells that express Lipoglobulin (i.e.,hepatocytes). In an additional embodiment, a vector capable ofexpressing Lipoglobulin or a preferred fragment may be administered to asubject to treat or prevent a condition associated with altered halflife, expression, or activity of the protein of the invention includingbut not limited to, those listed above.

In a still further embodiment, a vector of expressing the polynucleotideencoding Lipoglobulin or a fragment thereof may be administered to asubject to treat or prevent a condition associated with altered halflife, expression, or activity of the protein of the invention includingbut not limited to, those described above.

In another embodiment, the invention relates to a method of screeningfor compounds that bind a Lipoglobulin polypeptide or fragment thereof.Such method comprises the step of labeling the ligand molecule to betested with a detectable label, such as a fluorescent, radioactive, orenzymatic tag, placing the ligand molecule to be tested in contact withLipoglobulin polypeptide, or a fragment thereof under conditions whichpermit specific binding to occur, removing of non-specifically boundmolecules, and detecting bound molecules using appropriate means. In apreferred embodiment, the proteins of the invention or part thereof maybe used to identify and/or quantify substrates using any techniquesknown to those skilled in the art. To find substrates, the proteins ofthe invention, or part thereof, or derivative thereof, may be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, between theproteins of the invention, or part thereof, or derivative thereof, andthe agent being tested, may be measured by methods well known to thoseskilled in the art such as the BIAcore (Upsala, Sweden). Antagonists orinhibitors of the proteins of the invention may be produced usingmethods which are generally known in the art, including the screening oflibraries of pharmaceutical agents to identify those which specificallybind the protein of the invention. Another technique for drug screeningwhich may be used provides for high throughput screening of compoundshaving suitable binding affinity to the protein of the invention. Forexample, antagonists of Lipoglobulin polypeptide will prevent theinhibition of platelets and neutrophils by Lipoglobulin polypeptides.

Protein of SEQ ID NO:10 (Internal Designation Clone 164341117-001-5-0-E2-F)

The cDNA of clone 164341_(—)117-001-5-0-E2-F (SEQ ID NO:9) encodesperoxisomal beta ketothiolase (PBK) of SEQ ID NO:10, comprising theamino acid sequence:

MQRLQVVLGHLRGPADSGWMPQAAPCLSGAPQASAADVVVVHGRRTAICRAGRGGFKDTTPDELLSAVMTAVLKDVNLRPEQLGDICVGNVLQPGAGAIMARIAQFLSDIPETVPLSTVNRQCSSGLQAVASIAGWSPCPWLTEGTLEILLRA. Accordingly, it will beappreciated that all characteristics and uses of the polypeptides of SEQID NO:10 described throughout the present application also pertain tothe polypeptides encoded by the human cDNA included in Clone164341_(—)117-001-5-0-E2-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:9 describedthroughout the present application also pertain to the nucleic acidscomprising the human cDNA in Clone 164341_(—)117-001-5-0-E2-F. Apreferred embodiment of the invention is directed toward thecompositions of SEQ ID NO:9, SEQ ID NO:10, and clone164341-117-001-5-0-E2-F. Also preferred are polypeptide fragments havinga biological activity as described herein and the polynucleotidesencoding the fragments.

The protein of SEQ ID NO:10, PBK, is a splice variant of the sequence of3-Ketoacyl-CoA Thiolase protein (swissprot accession number P09110). The153 amino acid protein of PBK displays one membrane-spanning segment:CSSGLQAVASIAGWSPCPWLT (SEQ ID NO:61). Accordingly, some embodiments ofthe present invention relate to polypeptides comprising thetransmembrane domain. Finally, the protein of the invention displays athiolase domain spanning the sequence:APQASAADVVVVHGRRTAICRAGRGGFKDTTPDELLSAVMTAVLKDVNLRPEQLGDICVGNVLQPGAGAIMARIAQFLSDIPETVPLSTVNRQCSSGLQAVASIAGWSPCPWLTEGTLEIL (SEQ IDNO:62). Accordingly, a preferred embodiment of the present inventioncomprises the amino acids of the thiolase domain and polynucleotidesencoding the same.

Living organisms are exposed to a number of different fatty acids andtheir various derivatives arising either via endogenous synthesis orfrom exogenous sources. These hydrophobic compounds can have specificmetabolic, structural or endocrine functions before elimination, whichcan involve metabolism to CO₂ or to more polar lipid metabolitesallowing their excretion. Quantitatively, one of the major pathwaysmetabolizing fatty acids is beta-oxidation and PKB is involved in thispathway.

PBK is a ketoacyl-CoA thiolase involved in the formation of anacyl-enzyme intermediate and as such, plays a role in fatty acidmetabolism, cellular vesicle transport and maintenance of thecytoarchitecture, cellular proteolysis, endocytosis, signaltransduction, lysosomal storage, cell proliferation and differentiation,immune and inflammatory response. The enzyme's substrates are compoundspreferably containing an ester bond, preferably a thiol ester bond, morepreferably an acyl thioester bond.

PBK mediates lipid homeostasis in target organs like liver, adiposetissue and is regulated by peroxisome proliferator activated receptors(PPAR alpha and PPAR gamma). Deficiency of PBK leads to very lowperoxisomal beta-oxidation activity and the accumulation ofvery-long-chain fatty acids and intermediates in the biosynthesis ofbile acids. Such peroxisomal disorders are peroxisome-deficientZellweger syndrome and Rhizomelic Chondrodysplasia Punctata.

An additional preferred embodiment of the invention is a method ofbinding PBK polypeptides with a PBK-specific antibody or PBK-bindingfragment thereof. This method comprises the step of contacting a PBKpolypeptide with a PBK-binding antibody or PBK-binding fragment thereofunder conditions that allow binding. This method may be applied todetection and purification of PBK, as well as modification of PBKfunction. These aspects are discussed in detail herein.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a PBK polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing PBK expression.Preferably, the polynucleotides capable of directing PBK expression arelocated in the 5′ regulatory region of the PBK gene. Further preferably,these polynucleotides are located within 500 base pairs of the PBKcoding region. These polynucleotides preferably comprise a promotersequence. Techniques known in the art for introducing polynucleotidesequences to endogenous sequences are described in U.S. Pat. No.5,641,670 and PCT WO9629411, which disclosures are hereby incorporatedby reference in their entireties. PBK protein produced by said host cellmay be used for in vitro detection and purification methods as well asdiagnosis and in vivo applications.

In one embodiment, the hydrolytic activity of the protein of theinvention or part thereof may be assayed using any of the assays knownto those skilled in the art including those described in U.S. Pat. No.5,445,942, which disclosure is hereby incorporated in its entirety.

Another embodiment of the invention relates to compositions and methodsusing the protein of the invention or part thereof to label peroxisomes,in order to visualize any change in number, topology or morphology ofthis organelle, for example in association with beta-oxidationperoxisomal disorders such as, but not limited to Zellweger syndrome,Pseudo Zellweger, Rhizomelic Chondrodysplasia Punctata, X-linkedadrenoleukodystrophy (ALD), neonatal adrenoleukodystrophy,pseudoneonatal adrenoleukodystrophy, acyl-coA deficiency, bifunctionalenzyme deficiency, Refsum's disease, DHAP acyl transferase deficiency,hyperpipecolatemia and acatalasemia.

For example, the protein may be rendered easily detectable by insertingthe cDNA encoding the protein of the invention into a eukaryoticexpression vector in frame with a sequence encoding a tag sequence.Eukaryotic cells expressing the tagged protein of the invention may alsobe used for the in vitro screening of drugs or genes capable of treatingany beta-oxidation peroxisomal disorders.

An embodiment of the invention provides for a method of screening testsubstances for modulators of PBK expression. This method comprises thesteps of: i) contacting a cell with a test substance; and ii) comparingPBK expression in the cell after exposure to the test substance to thatof an unexposed control cell. PBK expression is determined by methodscommon to the art or included herein, by detecting PBK polynucleotidesor polypeptides. An example of this method comprises the steps of: i)culturing two equivalent cell samples; ii) adding a test substance toone of the cultures and not the other; iii) harvesting both cultures ata specified time; iv) purifying the mRNA from each sample of cells; v)comparing the level of PBK mRNA in each sample by Northern blot, RTPCR,or another method common to the art. The invention provides for designand use of specific polynucleotide probes and primers, as discussedherein. An additional example comprises the steps of: i) having twoequivalent cultures of cells; ii) adding a test substance to one of thecultures and not the other; iii) harvesting both cultures; iv) purifyingthe protein from each sample of cells; v) comparing the level of PBKpolypeptides in each sample by Western blot, immunohistochemistry, oranother method common to the art. The invention provides for design anduse of specific antibodies and antibody fragments, as discussed herein.

Agents which modulate the expression or activity of the PBK of thesubject invention include, but are not limited to antisenseoligonucleotides, ribozymes, drugs, and antibodies. These agents may bemade and used according to methods well known in the art. Also, agentswhich increase PBK expression are hypolipidemic compounds. Moreover, theprotein of the invention, or biologically active fragments thereof, maybe used in screening assays for therapeutic compounds. A variety of drugscreening techniques may be employed. In this aspect of the invention,the protein or biologically active fragment thereof, may be free insolution, affixed to a solid support, recombinantly expressed on, orchemically attached to, a cell surface, or located intracellularly. Theformation of binding complexes, between the protein of the invention, orbiologically active fragments thereof, and the compound being tested,may be measured by methods well known to those skilled in the art, like,but not limited to, the BIAcore (Upsala, Sweden). Another technique fordrug screening which may be used provides for high throughput screeningof compounds having suitable binding affinity to the protein of theinvention as described in published PCT application WO84/03564, andincorporated herein by reference in its entirety.

Another embodiment of the subject invention provides compositions andmethods of selectively increasing the activity of the protein of theinvention. Activation of PBK allows for the successful treatment and/ormanagement of diseases or biochemical abnormalities associated with PBKactivity. Agonists, able to increase the expression or the activity ofthe protein of the invention, are useful in the treatment of diseasesassociated with decreased peroximal beta-oxidation of fatty acids, forexample, Pseudo Zellweger syndrome and Rhizomelic ChondrodysplasiaPunctata (RCDP). Preferred agonists include hypolipidemic compounds.Each hypolipidemic compound may additionally be excluded individually oras a group.

In another embodiment, the present invention includes the use of PBKpolypeptides, or fragments having a desired biological activity to treator ameliorate a condition in an individual. For example, the conditionmay be Pseudo-Zellweger syndrome or Rhizomelic Chondrodysplasia Punctata(RCDP). In such embodiments, a PBK polypeptide or a fragment thereof, isadministered to an individual in whom it is desired to increase any ofthe activities of PBK. A PBK polypeptide or fragment thereof may beadministered directly to the individual or, alternatively, a nucleicacid encoding the a PBK polypeptide or a fragment thereof may beadministered to the individual. For example, a polynucleotide encoding aPBK polypeptide or a fragment is comprised in a recombinant expressionvector which can be administered to a patient suffering fromPseudo-Zellweger syndrome or Rhizomelic Chondrodysplasia Punctata(RCDP). Preferred expression vectors include viral vectors, especiallyadenoviral and lentiviral vectors. Alternatively, an agent whichincreases the activity of PBK polypeptides may be administered to theindividual. Preferred agonists are hypolipidemic compounds. Specifichypolipidemic compounds may additionally be excluded individually or asa group.

In such a method, an agent that increases PBK activity can be processedin accordance with conventional methods of pharmacy to produce medicinalagents for administration to patients. Thus, the pharmaceuticalcomposition comprising an agent that increases PBK activity may be madeup in a solid form (e.g. granules for oral administration, powders forinhalation) or in a liquid form (e.g. solutions for oral administrationor for injection). Effective doses of the compositions of the presentinvention for treating a patient suffering from disorders like, but notlimited to, Pseudo-Zellweger syndrome or Rhizomelic ChondrodysplasiaPunctata (RCDP), can be determined according to the relevant techniques.Additional agents may be identified by contacting a PBK polypeptide or acell or preparation containing a PBK polypeptide with a test agent andassaying whether the test agent increases the activity of the protein.For example, the test agent may be a chemical compound or a polypeptideor peptide.

In one embodiment, the subject method utilizes eukaryotic or prokaryotichost cells which are stably transformed with recombinant nucleic acidsexpressing the PBK polypeptide or biologically active fragments thereof.The transformed cells may be viable or fixed. Drugs or compounds whichare candidates for the activation of the protein of the invention, orbiologically active fragments thereof, are screened against suchtransformed cells in binding assays well known to those skilled in theart. Alternatively, assays such as those taught in Geysen H. N., WOApplication 84/03564, published on Sep. 13, 1984, and incorporatedherein by reference in its entirety, may be used to screen for peptidecompounds which demonstrate binding affinity for, or the ability toactivate, PBK polypeptides or biologically active fragments thereof.

Another embodiment of the invention relates to composition and methodsusing polynucleotide sequences encoding the protein of the invention orpart thereof to establish transgenic model animals (D. melanogaster, M.musculs), by any method familiar to those skilled in the art. Bymodulating in vivo the expression of the transgene with drugs ormodifier genes (activator or suppressor genes), animal models can bedeveloped that mimic human peroxisome-associated disorders such asZellweger syndrome, Rhizomelic Chondrodysplasia Punctata, X-linkedadrenoleukodystrophy (ALD), acyl-coA deficiency, bifunctional enzymedeficiency, Refsum's disease, DHAP acyl transferase deficiency andacatalasemia. These animal models would thus allow the identification ofpotential therapeutic agents for treatment of the disorders. Inaddition, recombinant cell lines derived from these transgenic animalsmay be used for similar approaches ex vivo.

In another embodiment, the invention relates to methods and compositionsfor detecting and quantifying the level of the protein of the inventionpresent in a particular biological sample. These methods are useful forthe diagnosis or prognosis of diseases associated with altered levels ofthe protein of the invention like, but not limited to, pseudo-Zellwegersyndrome. Diagnostic assays to detect the protein of the invention maycomprise a biopsy, in situ assay of cells from organ or tissue sections,or an aspirate of cells from a tumor or normal tissue. In addition,assays may be conducted upon cellular extracts from organs, tissues,cells, urine, or serum or blood or any other body fluid or extract.

Assays for the quantification of the protein PBK may be performedaccording to methods well known in the art. Typically, these assayscomprise contacting the sample with a ligand of the protein of theinvention or an antibody (polyclonal or monoclonal) which recognizes theprotein of the invention or a fragment thereof, and detecting thecomplex formed between the protein of the invention present in thesample and the ligand or antibody. Fragments of the ligands andantibodies may also be used in the binding assays, provided thesefragments are capable of specifically interacting with the protein ofthe subject invention. Further, the ligands and antibodies which bind tothe protein of the invention may be labeled according to methods knownin the art. Labels which are useful in the subject invention include,but are not limited to, enzymes labels, radioisotopic labels,paramagnetic labels, and chemiluminescent labels. Typical techniques aredescribed by Kennedy, J. H., et al. (1976) Clin. Chim. Acta 70:1-31,which disclosure is hereby incorporated by reference in its entirety;and Schurs, A. H. et al. (1977) Clin. Chim. Acta 81: 1-40, whichdisclosure is hereby incorporated by reference in its entirety). Forexample, PBK will be detected in fibroblasts of control individuals butnot of Zellweger patients by immunoblot.

In another embodiment, an array of oligonucleotides probes comprisingthe nucleotides encoding PBK polypeptidesor fragments thereof can beconstructed to conduct efficient screening of e.g., genetic mutations. Amicroarray can be used to monitor the expression level of large numbersof genes simultaneously and to identify genetic variants, mutations, andpolymorphisms. This information may be used to determine gene function,to understand the genetic basis of a disorder, to diagnose a disorder,and to develop and monitor the activities of therapeutic agents (see forexample: Chee, M. et al., Science, 274:610-614 (1996), which disclosureis hereby incorporated by reference in its entirety). Genetic variants,mutations, and polymorphisms of PBK gene are related to peroxisomalbeta-oxidation disorders such as Rhizomelic Chondrodysplasia Punctata.

Protein of SEQ ID NO:12 (Internal Designation Clone 1000837037228-43-2-0-C3-F)

The cDNA of Clone 1000837037_(—)228-43-2-0-C3-F (SEQ ID NO: 11) encodesmyeloidin of SEQ ID NO:12, comprising the amino acid sequence:MPFSHLSTYSLVWVMAAVVLCTAQVQVVTQDEREQLYTTASLKCSLQNAQEALIVTWQKKKAVSPENMVTFSENHGVVIQPAYKDKINITQLGLQNSTITFWNITLEDEGCYMCLFNTFGFGKISGTACLTVYVQPIVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLHLGTVTDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILLYWKRHRNQDREP. Accordingly, it will be appreciated that allcharacteristics and uses of the polypeptides of SEQ ID NO:12 describedthroughout the present application also pertain to the polypeptidesencoded by the human cDNA included in Clone1000837037_(—)228-43-2-0-C3-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:11described throughout the present application also pertain to the nucleicacids comprising the human cDNA included in Clone1000837037_(—)228-43-2-0-C3-F. A preferred embodiment of the inventionis directed toward the compositions of SEQ ID NO:12, SEQ ID NO:11, andClone 1000837037_(—)228-43-2-0-C3-F. Preferred myeloidin polypeptidesfor uses in the methods described below include the polypeptidescomprising the amino sequence of:QVQVVTQDEREQLYTTASLKCSLQNAQEALIVTWQKKKAVSPENMVTFSENHGVVIQPAYKDKINITQLGLQNSTITFWNITLEDEGCYMCLFNTFGFGKISGTACLTVYVQPIVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLHLGTVTDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILLYWKRHRNQDREP (SEQ ID NO:127).Also preferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The protein of the present invention, myeloidin, is a novel splicevariant of the brain OX-2 protein (Genbank accession number P41217).Myeloidin is 262 amino acids long. OX-2 is 274 amino-acids long anddiffers from myeloidin at both the amino-terminal and thecarboxyl-terminal extremities. Another known splice variant of OX-2,my033, is 269 amino acids long and differs from myeloidin at theamino-terminal extremity. Myeloidin displays a signal peptide(MPFSHLSTYSLVWVMAAVVLCTA) (SEQ ID NO:63), one transmembrane domain(VPLLLSIVSLVILLVLISILLYW) (SEQ ID NO:64) and two immunoglobulin (Ig)domains (TASLKCSLQNAQEALIVTWQKKKAVSPENMVTFSENHGVVIQPAYKDKINITQLG LQNSTITFWNITLEDEGCYMCLF (SEQ ID NO:65) and EDHLNITCSATARPAPMVFWKVPRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQV (SEQ ID NO:66)). Myeloidin isexpressed by a wide variety of cells, including those of the centralnervous system (CNS).

Myeloidin is a cell surface protein that delivers an inhibitory signalfor myeloid lineage cells. Myeloidin binds to a receptor specificallylocated on myeloid lineage cells. In peculiar, myeloidin delivers aninhibitory signal to brain microglial cells. Microglial cells regulatethe regenerative state and remodeling of the brain by producing avariety of cytotoxic and neurotrophic molecules when activated. Moregenerally, myeloidin is involved in immune suppression by inhibitingcytotoxic cells.

An embodiment of the invention is directed to a composition comprising amyeloidin polypeptide sequence of SEQ ID NO:12.

A further embodiment of the invention is directed to a compositioncomprising a myeloidin polypeptide fragment having biological activityof delivering an inhibitory signal for myeloid lineage cells.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO: 11 encoding amyeloidin polypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a myeloidin polypeptidefragment having biological activity of delivering an inhibitory signalfor myeloid lineage cells.

A further embodiment of the invention is directed to a compositioncomprising an antibody recognizing a myeloidin polypeptide sequence ofSEQ ID NO: 2 or a myeloidin polypeptide fragment having same biologicalactivity. Preferably, the antibody binds to myeloidin but not to OX-2 orto my033. As further used herein, an anti-myeloidin antibody refers toan antibody that specifically binds either to a myeloidin polypeptide orto a myeloidin polypeptide fragment having the same biological activity.An anti-myeloidin antibodies may be an entire molecule or an antigenbinding fragment thereof.

The present invention also relates to a method of binding ananti-myeloidin antibody to a myeloidin polypeptide comprising the stepof: contacting a myeloidin polypeptide with said antibody underconditions that allow binding to take place. Such conditions are wellknown to those skilled in the art. Additionally, the present inventionalso relates to a method of binding an anti-myeloidin antibody to a cellexpressing a myeloidin polypeptide or fragment thereof comprising thestep of: contacting a cell expressing a myeloidin polypeptide or partthereof with said antibody under conditions that allow binding to takeplace. Such methods of binding an anti-myeloidin antibody to a myeloidinpolypeptide are for example useful for purifying myeloidin polypeptides.

Another embodiment relates to a method of producing myeloidinpolypeptides comprising the steps of: i) transfecting a host cell with arecombinant expression vector comprising a polynucleotide of the presentinvention, and optionally ii) purifying the produced protein. Thepurification of the protein can be done following any techniquewell-known to those skilled in the art. Preferably, an anti-myeloidinmay be bound to a chromatographic support to form an affinitychromatography column. Alternatively, myeloidin polypeptides may beproduced by a method comprising the step of: i) transfecting a host cellwith a polynucleotide capable of directing myeloidin expression.Preferably, the polynucleotides capable of directing myeloidinexpression are located in the 5′ regulatory region of the myeloidingene. Further preferably, these polynucleotides are located within 500base pairs of the myeloidin coding region. These polynucleotidespreferably comprise a promoter sequence. Techniques known in the art forintroducing polynucleotide sequences to endogenous sequences aredescribed in U.S. Pat. No. 5,641,670 and PCT WO9629411, whichdisclosures are hereby incorporated by reference in their entireties.

An additional aspect of the previous embodiment relates to methods ofproducing myeloidin in vivo in a mammal. Preferred such method is amethod of genetically modifying donor cells by gene transfer ofmyeloidin polynucleotides for grafting the cells into the centralnervous as described in U.S. Pat. No. 5,762,926, which disclosure isincorporated by reference in its entirety. Preferably, donor cells areneurons or fibroblasts. Most preferably, such methods are applied to amammal suffering from neurodegenerative disorders. Preferred mammals aremice and humans.

One embodiment of the present invention is directed to methods ofisolating or purifying microglial cells using myeloidin polypeptides.The study and use of isolated microglial cells and purified microglialcell populations (including, for example, characterizing the interactionthese cells in vitro or in vivo with drugs and drug candidates) providesinformation useful in treating neurological disorders. One method forobtaining a cell population enriched in microglial cells comprises thesteps of: i) contacting a preparation comprising microglial cells with amyeloidin polypeptide, wherein myeloidin is immobilized on a matrixprior to or after contacting; and ii) removing non-adherent cells,thereby producing cell population enriched in microglial cells. Forexample, the matrix may be plastic and the myeloidin polypeptide may beimmobilized by adsorption. Another method for isolating or purifyingmicroglial cell populations using compositions comprising using anmyeloidin polypeptide comprises the steps of: i) labeling by standardmethods the myeloidin polypeptide with a molecule that can be used toprovide a detectable signal, ii) contacting the labeled myeloidinpolypeptide with a cell under conditions that allow protein binding, andiii) sorting the cell based on the presence or absence of the detectablesignal. Sorting and detection are preferably accomplished by a sortingapparatus, e.g. a fluorescence-activated cell sorter or a magneticactivated cell-sorting apparatus. Preferably, the preparationscomprising microglial cells are prepared from brain (e.g., from rat ormouse brain) and may be from a normal mammal or from a mammal with aneurological disorder. Preparation of cell suspension tissue containingmicroglia can be performed according to any method well-known to thoseskilled in the art, e.g., the methods described in PCT application WO01/51618, which disclosure is hereby incorporated by reference in itsentirety.

Another preferred embodiment relates to methods of screening forsubstances that increase or decrease myeloidin expression comprising thesteps of: i) contacting a cell with a test substance; and ii) comparingmyeloidin expression in the cell after exposure to the test substance tothat of an unexposed control cell. Preferably, the test substancemodifies the expression of myeloidin in a specific cell type while notin others. Most preferably, the test substance modifies myeloidinexpression specifically in neural cells.

A further embodiment of the present invention is directed to substancesthat increase or decrease myeloidin activity and to a method ofscreening for such substances comprising the steps of: i) contacting acell with a test substance, ii) determining myeloidin activity, iii)comparing myeloidin activity in the cell after exposure to that of anunexposed control cell, and iv) determining the ratio of myeloidinactivity in an exposed cell relative to the activity in an unexposedcell. Preferably, myeloidin activity is studied in a neural cell.Myeloidin activity can be determined by studying microglial-mediatedactivation of astrocytes, i.e., to determine its ability to prevent areactive astrocyte morphology in culture. Myeloidin activity can also bedetermined by measuring cytokine production as described in PCTapplication WO 99/24565, which disclosure is hereby incorporated byreference in its entirety.

As further used herein, substances that increase myeloidin expression ormyeloidin activity are defined as myeloidin agonists. As further usedherein, substances that decrease myeloidin expression or myeloidinactivity are defined as myeloidin antagonists. The term antagonistcomprises anti-myeloidin antibodies that inhibit the biological activityof myeloidin polypeptides.

An embodiment of the present invention relates to methods of usingmyeloidin antagonists of the present invention to induce or to enhanceactivation of myeloid lineage cells. Such methods comprise the step of:contacting a cell, a tissue, or an individual with a compositioncomprising a myeloidin antagonist and optionally a pharmaceuticalcarrier. In a preferred embodiment, such methods for activating myeloidlineage cells are directed toward brain microglial cells. Such methodscan be used to destroy invading microorganisms, to promoteneuroregeneration, or to remove potentially deleterious debris andpromote tissue repair consecutively to brain injury.

Infections that can be treated or reduced in severity by activatingmicroglial cells include but are not limited to brain abscess due to,e.g., Aspergillus CNS infections, meningitis due to, e.g., CryptococcusCNS infections, neuropsychiatric disorders associated with streptococcalinfection (PANDAS), and influenza virus-associated encephalopathy.

Another embodiment of the present invention relates to methods of usingthe polypeptides and the polynucleotides of the present invention toprevent or to reduce activation of myeloid lineage cells. Such methodscomprise the step of: contacting a cell, a tissue, or an individual witha composition comprising a myeloidin polypeptide or a myeloidin agonist,and optionally a pharmaceutical carrier. In a preferred embodiment, suchmethods for preventing or reducing myeloid lineage cell activation aredirected toward brain microglial cells. Such methods can be used totreat or to reduce in severity neural inflammation, and morespecifically neurodegenerative disorders and pathological inflammationsconsecutive to brain injury. Neurodegenerative disorders associated withneural inflammation include but are not limited to stroke, Parkinson'sdisease, Alzheimer's disease, cerebellum-type Creutzfeldt-Jakob disease,and Huntington's disease.

In another preferred embodiment, such methods for preventing or reducingmyeloid lineage cell activation are directed for preventing or treatinginflammation associated with autoimmune diseases. Autoimmune diseasesthat may be treated or prevented according to the present inventioninclude, but are not limited to, multiple sclerosis, type 1insulin-dependant diabetes mellitus, and rheumatoid arthritis.

Effectiveness of compositions and methods of the present invention tomodulate microglial activity can be verified in vitro by studying theeffects of the compositions of the present invention on the morphologyof astrocytes. Effectiveness of compositions and methods of the presentinvention to treat a given disease associated with microglial activationcan be verified using models that depend upon the disease that istreated. For example, one model described in U.S. Pat. No. 6,191,154,which disclosure is hereby incorporated by reference in its entirety,can be used as a tissue model for the sequence of events followingtrauma in the nervous system.

The quantity of myeloidin polypeptides, anti-myeloidin antibodies,myeloidin agonists or myeloidin antagonists that is administered fortreating a patient suffering from diseases described above can bedetermined according to the particular application and the potency ofthe active component. For preparing pharmaceutical compositions from thecompounds of the present invention, pharmaceutically acceptable carrierscan be in any suitable form (e.g., solids, liquids, gels). The presentinvention contemplates a variety of techniques for administration of thetherapeutic compositions. Suitable routes include, but are not limitedto, oral, rectal, or transdermal administration, and intradermal,subcutaneous or intravenous injections. Indeed, it is not intended thatthe present invention is limited to any particular administration route.The assessment of the clinical features and the design of an appropriatetherapeutic regimen for the individual patient are ultimately theresponsibility of the prescribing physician. Furthermore, thecompositions of the invention may be administered alone or incombination with other known agents treating the disorders treatedabove.

Protein of SEQ ID NO:14 (Internal Designation Clone 101005105-020-4-0-H1-F)

The cDNA of Clone 101005_(—)105-020-4-0-H11-F (SEQ ID NO:13) encodesprotein vACT of SEQ ID NO:14, comprising the amino acid sequence:MERMLPLLTLGLLAAGFCPAVLCIHPNSPLDEENLTQENQDRGTHVDLGLASANVDFALSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAKWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTVVELKYTGNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISRDYNLNDILLQLGIEEAFTSKADLSGITGARNLAVSQVVHKAVLDVFEEGTEASAATAVKITLLSALVETRTIVRFNRPFLMIIVPTDTQNIFFMSKVTNPKQA. Accordingly, it will beappreciated that all characteristics and uses of polypeptides of SEQ IDNO:14 described throughout the present application also pertain to thepolypeptides encoded by the human cDNA in Clone101005_(—)105-020-4-0-H11-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:13described throughout the present application also pertain to the nucleicacids comprising the human cDNA in Clone 101005_(—)105-020-4-0-H11-F. Apreferred embodiment of the invention is directed toward thecompositions of SEQ ID NO:13, SEQ ID NO:14, and Clone101005_(—)105-020-4-0-H1-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

The cDNA of SEQ ID NO:13 is a novel polymorphic variant of the humanalpha 1 anti-chymotrypsin (ACT) protein named vACT, encoded by a genelocated on chromosome 14, specifically at position 14q31-q32.3. The cDNAof SEQ ID NO:13 encodes a 423 amino-acid protein of SEQ ID NO:14.

Proteases are key components of a broad range of biological pathways andcan be classified into four groups according to their catalyticmechanisms: the serine, cysteine (thiol), aspartic (carboxyl), andmetalloproteases. Chymotrypsin is a member of a family of enzymes knownas serine proteases, so named because they have an unusually reactiveserine residue at their active sites. vACT belongs to the serpin serineprotease inhibitor family. Serpins are irreversible suicide inhibitorsof proteases that have a central role in regulating proteolysis indiverse physiological processes such as blood coagulation, fibrinolysis,complement activation, angiogenesis, apoptosis, inflammation, neoplasiaand viral pathogenesis. vACT neutralizes chymotrypsin by binding to itsactive site and forming a stable complex. vACT is predominantlysynthesized in the liver and specifically inactivates neutrophilcathepsin G, mast cell chymase and pancreatic chymotrypsin. vACT is alsoan acute phase protein since the plasma concentration of vACT increaseswithin several hours during the inflammatory response. Synthesis of vACTis tightly regulated by the net balance of neutrophil cathepsin G andvACT at sites of inflammation/tissue injury. Alterations of a serpinwhich affect its functional levels may result in pathology and causespecific clinical syndromes. For example, individuals with vACTdeficiency are susceptible to premature development of lung and liverdiseases. In addition, changes in the balance between serine proteasesand vACT may lead to pathological states similar to those associatedwith some neurodegenerative diseases such as Alzheimer's disease.

An embodiment of the invention is directed to a composition comprising avACT polypeptide sequence of SEQ ID NO:14.

A further embodiment of the invention is directed to a compositioncomprising a vACT polypeptide fragment having biological activity.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:13 encoding a vACTpolypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a vACT polypeptidefragment having biological activity.

In an embodiment, a vACT polypeptide or fragment thereof may be used toinhibit contaminating proteases in a sample. This method comprises thestep of adding a protease-inhibiting amount of vACT polypeptide tobiological samples, under conditions that allow vACT activity to preventdegradation of protein samples. Preferred biological samples are cellcultures. Such a composition can be used alone or as a “cocktail” withother protease inhibitors. The advantage of using a cocktail of proteaseinhibitors is that one is able to inhibit a wide range of proteaseswithout knowing the specificity of any of the proteases. Using acocktail of protease inhibitors also protects a protein sample from awide range of proteases which may contaminate a protein sample in thefuture from a vast number of sources. For example, vACT polypeptides orfragments thereof are added to samples where proteolytic degradation bycontaminating proteases is undesirable. Such protease inhibitorcocktails are widely used in assays to inhibit proteases susceptible ofdegrading a protein of interest for which the assay is to be performed.Alternatively, vACT or part thereof may be bound to a chromatographicsupport, either alone or in combination with other protease inhibitor,using techniques well known in the art, to form an affinitychromatography column. A sample containing the undesirable protease isrun through the column to remove the protease. Alternatively, the samemethods may be used to identify new target proteases of vACT.

In another preferred embodiment, vACT polypeptides or fragments thereofmay be used as an anti-microbial agent useful to inhibit exogenousproteases implicated in a number of infectious diseases including, butnot limited to, bacterial and parasite-borne infections. For example,vACT inhibits growth of all strains of group A streptococci, includingantibiotic-resistant strains. Accordingly, the present invention can beused to retard or inhibit the growth of certain microbes either in vitroor in vivo. Such methods comprise the step of delivering an amount ofvACT polypeptide able to inhibit serine proteases, including byadministering purified protein or transfecting cells with apolynucleotide encoding the protein. In vitro, this method comprises thestep of contacting an effective amount of vACT polypeptide with abiological sample. Preferred biological samples are cell cultures. Invivo, this method comprises the step of contacting an effective amountof vACT polypeptide with a desired site in an individual. Preferredindividuals are those at risk of microbial infection.

In a further embodiment, the present invention provides a method ofproducing a recombinant serpin capable of effectively modulating serineprotease activity. Despite the availability of human alpha 1anti-chymotrypsin and vACT from serum, quantities large enough fortherapeutic uses have been unobtainable, due in large part to thelimited availability of human serum. Consequently, there is a great needfor other sources of alpha 1 anti-chymotrypsin and vACT to fill theneeds created by therapeutic uses. In one preferred embodiment, a milkanimal can be used to produce vACT in the milk, thereby generating asignificant amount of this particular protein after purification. Theprotein of the invention may be purified using any techniques known tothose skilled in the art including those disclosed in the U.S. Pat. No.6,268,487, which disclosure is hereby incorporated by reference in itsentirety. Any type of animal that produces enough milk can be used inthis aim such as, but not limited to, sheep, goat, and cow. Theseanimals can be generated with any method of targeting overexpression ofvACT in the milk. Also in this embodiment, the protein of the inventioncan be produced in host cells that have been transfected with anappropriate expression vector comprising a nucleic acid sequence codingfor vACT. The host cells are cultured under conditions whereby thenucleic acid sequence coding for this particular protein is expressed.After a suitable amount of time for the product to accumulate, theprotein is purified from the host cells or medium surrounding the cells.Introduction of an expression vector incorporating a nucleic acidsequence coding for vACT into a host cell can be performed in a varietyof ways, such as but not limited to calcium or lithium chloridetreatment, electroporation, and lipofection.

The present invention also provides animal models generated bymodulating the expression or activity of the present protein in one ormore tissues of the animal. Such animals are useful for a number ofpurposes, because they represent an in vivo assay method for testingcandidate molecules potentially useful for the treatment of variouspathophysiological aspects of diseases specifically related to theactivity of vACT. Study of the phenotype of such models can also allowthe identification of additional human equivalent diseases caused by orlinked with vACT deficiency. These animals can be generated with anymethod of targeting overexpression or inactivation of vACT. Such modelsare extremely useful, e.g. in the assessment of candidate therapies anddrugs for the treatment of inflammatory diseases and conditions.

A further embodiment of the present invention is to provide novelmethods and compositions useful for the treatment of diseases andconditions exhibiting excessive activity of serine proteases andpreferably chymotrypsin. vACT or part thereof may be used to inhibitproteases implicated in a number of diseases where cellular proteolysisoccurs. Such diseases, characterized by tissue degradation, include, butare not limited to, blood coagulation-related diseases, tumor invasion,infection, and inflammation. More preferably, the present invention isapplied in the treatment of diseases associated with an excess level ofcathepsin G, mast cells chymase or pancreatic chymotrypsin including butnot limited to, chronic emphysema of the lungs, liver diseases,pancreatitis, cardiovascular diseases, and allergic reactions. Themethods and compositions are also useful for treatment of Alzheimer'sand Parkinson's diseases. Such methods comprise the administration of atherapeutically-effective amount of vACT to mammals suffering from thedisease or condition, where “effective amount” is meant a concentrationof vACT capable of significantly decrease the activity of serineproteases. The compositions of the invention are preferably delivered toan individual in combination with a physiologically acceptable carrier,such as a saline solution or other physiologically buffer suitable foradministration to a patient. For treatment of skin inflammation, thecompositions of the invention may be applied to the affected area incombination with a physiologically acceptable ointment or cream. Theparticular amount of the compositions of the invention that will beadministered to the mammal for any particular condition will depend onthe clinical condition of the patient, and other factors such as theweight, age, and route of delivery. Such compositions can beadministered by any suitable route including, but not limited to,intravenous, intramuscular, intraperitoneal, subcutaneous routes, andtopically to an affected area of the skin or by absorption throughepithelial or mucocutaneous linings such as nasal, oral, vaginal,rectal. For treatment purposes, the compositions may be administratedusing any of the gene therapy methods known in the art to deliver thetherapeutic bioactive molecules to targeted cells. These compositionscan comprise the protein of the invention, and, optionally, one or moreother types of protease inhibitors, or any other compound of interest.This co-administration may be by simultaneous administration or byseparate or sequential administrations. All of these additionalcomponents may be either obtained from natural sources or produced byrecombinant genetic engineering techniques and/or chemical modification.

An additional preferred embodiment of the invention is a method ofbinding vACT polypeptides with a vACT-specific antibody or vACT-bindingfragment thereof. This method comprises the step of contacting a vACTpolypeptide with a vACT-binding antibody or vACT-binding fragmentthereof under conditions that allow binding. This method may be appliedto detection and purification of vACT, as well as modifying vACTfunction. These aspects are discussed in detail herein.

In other embodiment, the current invention is used to diagnose diseasesor disorders associated with altered expression or activity of thepresent protein. In particular, it is useful in diagnosing patients withabnormally low levels of vACT expression, which results in uncontrolledactivity of target proteases. Examples of such diseases and disordersinclude, but are not limited to, lung and liver diseases, chronicobstructive pulmonary disease (COPD), inflammatory disorders associatedwith an excess level of cathepsin G, and Alzheimer's and Parkinson'sdiseases. The method includes the steps of: i) contacting a fluid ortissue sample with a compound capable of selectively binding vACTpolypeptide or nucleic acids, and ii) detecting the level, or any otherdetectable property of vACT in the sample. Preferably, a difference inthe level or other property in the sample relative to in a controlsample indicates the presence of the disease or disorder, or of apropensity for developing the disease or disorder. Preferably, the fluidor tissue sample is obtained from an individual suspected of sufferingfrom the disease or condition, or at risk of developing the disease orcondition. For example, a polyclonal or monoclonal antibody or anyimmunologically active fragment thereof or a nucleic acid probe may beused. Preferably, the antibodies used in one such embodiment arespecifically directed against the variant vACT polypeptides and do notrecognize alpha 1 anti-chymotrypsin polypeptides. Detection can becarried out directly or indirectly with known immunohistological andimmunofluorescing processes. For this, the reagents contained in the kitaccording to the invention can be directly labeled with generally knownmolecules, including, but not limited to, enzymes such as alkalinephosphatase and peroxidase and fluorescent dyes such as FITC, rhodamine,and Texas-Red. However, labeling can also occur indirectly by usingsecondary antibodies labeled with molecules such as biotin, digoxigeninor the like and are then detected with a secondary reagent. Also in thisembodiment, diagnosis of such conditions may be facilitated by theidentification of the variant vACT using well known PCR or RT-PCRtechniques and in particular in with “real-time” PCR system.Alternatively, using such a method, the present invention provides atool to correlate modulations in the expression of vACT with certainpathologies. Thus, the present invention provides a novel candidate genefor such conditions.

Since the regulation of serine proteases by their inhibitors arecritical for the control of tissue destruction in the diseases describedabove, in a further embodiment, the present protein or part thereofprovides an assay for the monitoring of markers in vivo forcharacterization of disease states. The invention thus includes testkits useful for the quantification of the amount of vACT in a biologicalsample. The kits comprise at least one immunological binding partner,e.g. a monoclonal or polyclonal antibody specific for vACT and coupledto detectable markers. Preferably, the antibodies used in one suchembodiment specifically bind vACT polypeptides and not alpha 1anti-chymotrypsin polypeptides. In this embodiment, the application ofsuch assays can be used to monitor the progress of therapy administeredto treat these or other conditions. Further, the assays can be used as ameasure of toxicity, or during clinical testing of new drugs to assessthe impact on tissue degradation. Thus the assays may be applied in anysituation wherein the present invention can be used as an index of thecondition, treatment, or effect of substances directly administered tothe subject or to which the subject is exposed in the environment. Thus,the condition of a patient can be monitored continuously and thequantified amount of such proteins measured in the pathological samplecan be compared with the amount quantified in a biological sample of anormal individual or with the previous analysis of the same patient. Inthis embodiment, this marker can be measured effectively in plasma,serum or blood, by any suitable method, including immunoassays.

It can also preferably be measured in tissues and fluids recovered frominflammatory sites.

Protein of SEQ ID NO:16 (Internal Designation 500743419188-281-3-0-H5-F)

The cDNA of 500743419_(—)188-281-3-0-H5-F (SEQ ID NO:15) encodes proteinclaudinyn-5 of SEQ ID NO:16, comprising the amino acid sequence:MGSAALEILGLVLCLVGWGGLILACGLPMWQVTAFLDHNIVTAQTTWKGLWMSCVVQSTGHMQCKVYDSVLALSTEVQAARALTVSAVLLAFVALFVTLAGAQCTTCVAPGPAKARVALTGGVLYLFCGLLALVPLCWFANIVVREFYDPSVPVSQKYELGAALYIGWAATALLMVGGCLLCCGAWVCTGRPDLSFPVKYSAPRRPTATGDNDKKNYV. Accordingly, it will beappreciated that all characteristics and uses of polypeptides of SEQ IDNO:16 described throughout the present application also pertain to thepolypeptides encoded by the nucleic acids included in500743419_(—)188-281-3-0-H5-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:15described throughout the present application also pertain to the nucleicacids comprising the human cDNA in 500743419_(—)188-281-3-0-H5-F. Apreferred embodiment of the invention is directed toward thecompositions of SEQ ID NO:15, SEQ ID NO:16, and500743419_(—)188-281-3-0-H5-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

The cDNA of SEQ ID NO:15 is a polymorphic variant of the human claudin-5protein belonging to the PMP22-Claudin family of proteins characterizedby four membrane-spanning segments. Claudinyn-5 is 218 amino acids longand contains four membrane-spanning segments. Claudinyn-5 contains theClaudin family signature and is encoded by a gene located on chromosome22, specifically at position 22q11.2.

The Claudin family of proteins comprises more than twenty smallglycoproteins with four predicted transmembrane domains. Claudinyn-5 isa component of tight junction (TJ) strands that play a role inregulation of cell permeability and polarity. Polarized epithelial andendothelial cells form barriers that separate biological compartmentsand regulate homeostasis. The TJ is a specialized membrane domain at themost apical region of polarized epithelial and endothelial cells thatconstitute continuous seals around cells. These seals serve as aphysical barrier preventing solutes and water from passing freelythrough the paracellular space. The TJ also restricts the lateraldiffusion of membrane lipids and proteins to maintain the cellularpolarity. Many diseases marked by changes in cell barrier permeabilityare in turn related to alteration in TJ function. For example, theincrease in microvascular permeability in tumors, contributing toclinically severe symptoms, appears to be the result of a dysregulationof junctional proteins. Increased TJ permeability of the epithelium, andconsequently a decrease in epithelial barrier function, precedes thedevelopment of some tumors, including carcinomas and adenomas. The lungepithelium forms a barrier that allergens must cross before they cancause sensitization. The nonspecific disruption of intercellular TJs byallergens increases epithelial permeability, allowing allergens to crossthe epithelial barrier. Finally, TJ permeability can also be modified bydifferent bacterial toxins, cytokines, hormones and drugs.

An embodiment of the invention is directed to a composition comprising aclaudinyn-5 polypeptide sequence of SEQ ID NO:16.

A further embodiment of the invention is directed to a compositioncomprising a claudinyn-5 polypeptide fragment having biologicalactivity.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:15 encoding aclaudinyn-5 polypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a claudinyn-5 polypeptidefragment having biological activity.

A further embodiment of the invention is directed to a compositioncomprising an antibody recognizing a claudinyn-5 polypeptide sequence ofSEQ ID NO:16 or a claudinyn-5 polypeptide fragment having biologicalactivity. Preferably, the antibody recognizes a non-linear epitopes, andbinds to claudinyn-5 but not to claudin-5.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a claudinyn-5 polypeptide or a biologicallyactive fragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing claudinyn-5expression. Preferably, the polynucleotides capable of directingclaudinyn-5 expression are located in the 5′ regulatory region of theclaudinyn-5 gene. Further preferably, these polynucleotides are locatedwithin 500 base pairs of the claudinyn-5 coding region. Thesepolynucleotides preferably comprise a promoter sequence. Techniquesknown in the art for introducing polynucleotide sequences to endogenoussequences are described in U.S. Pat. No. 5,641,670 and PCT WO9629411,which disclosures are hereby incorporated by reference in theirentireties. Claudinyn-5 protein produced by said host cell may be usedfor in vitro detection and purification methods as well as diagnosis andin vivo applications.

Another embodiment relates to a method of producing claudinyn-5polypeptides comprising the steps of: i) transfecting a mammalian hostcell with a recombinant expression vector comprising a polynucleotide ofthe present invention, and ii) purifying the produced protein. Thepurification of the protein can be done following any techniquewell-known to those skilled in the art. Preferably, an antibody directedagainst claudinyn-5 or part thereof may be bound to a chromatographicsupport to form an affinity chromatography column. Even more preferably,the antibodies bind to claudinyn-5 but not to claudin-5.

In another embodiment, the present invention provides for a method toincrease the intestinal absorption of hydrophilic drugs by increasingthe paracellular permeability for said drugs either in vitro or in vivo.Indeed, the intestinal epithelium is a major barrier to the absorptionof hydrophilic drugs. The presence of intercellular junctionalcomplexes, and particularly the TJs, renders the epithelium imperviousto hydrophilic drugs, which cannot diffuse across the cells through thelipid bilayer of the cell membranes. The expression of claudinyn-5 canbe inhibited or decreased for example using antisense_polynucleotidescorresponding to claudinyn-5 to down-regulate expression of saidproteins. Methods of designing, synthesizing, and using said antisensepolynucleotides are well known to those skilled in the art and arediscussed herein. The function of claudinyn-5 in TJ complexes can alsobe more specifically inhibited using direct or indirect inhibitormolecules or antagonistic antibodies directed against the presentprotein. Any compound that inhibits or significantly decreases theactivity of claudinyn-5 polypeptides can be identified by screening fortest substances that decrease claudinyn-5 activity comprising the stepsof contacting a cell with a test substance and comparing claudinyn-5activity in the cell after exposure to the test substance to that of anunexposed control cell. Preferably, claudinyn-5 activity is determinedby measuring the permeability of the TJs using, for example, methodsdiscussed in U.S. Pat. No. 6,110,747, which disclosures are herebyincorporated by reference in its entirety. Preferably, such methods areused is to temporally increase epithelial permeability for therapeuticends including but not limited to increasing the efficiency of drugdelivery. More preferably, for in vivo purposes, formulations are thosecomprising said compositions that are compatible with oral delivery.

In a further embodiment, a claudinyn-5 polypeptide-specific antibody ora fragment thereof can be used as a component of drug delivery vehiclessuch as colloids or liposomes to specifically target therapeutic agentsto epithelial cells. Such methods comprise the step of incorporating aclaudinyn-5 polypeptide-specific antibody or a fragment thereof, intoliposomes used to specifically target therapeutic agents carried by theliposomes to epithelial cells. Such drug delivery systems can bedesigned using any techniques known to those skilled in the art. In onesuch embodiment, claudinyn-5-specific antibodies or fragments thereofcan also be directly conjugated (either recombinantly or by usingchemically active agents) to chemotherapeutic agents, radioisotopes, orprodrugs and said conjugate subsequently used in therapeutic regimens.

The negative effects of the usual preservation solutions on epithelialand endothelial permeability in organs to be transplanted are generallyknown, considering the disorganization of TJ proteins. This isresponsible for the observed tissue injury and edema. In anotherembodiment, agonists of the expression of claudinyn-5 can be used tomaintain the content and integrity of TJs in organs to be transplanted.Further preferred embodiment is a method of using such agonists asconstituents of the preservation solutions in order to improve thequality of said organs to be transplanted. Any compound that stimulatesor significantly increases the expression of claudinyn-5 polypeptidescan be used in one such embodiment. Such compounds can be identified byscreening for test substances that increase claudinyn-5 expressioncomprising the steps of contacting a cell with a test substance andcomparing claudinyn-5 expression in the cell after exposure to the testsubstance to that of an unexposed control cell. In this embodiment, theactivity of claudinyn-5 polypeptides can also be increased to preserveTJ function by administering a compound that causes an increase in theactivity of the protein. Such compounds can be identified by screeningfor test substances that increase claudinyn-5 activity comprising thesteps of contacting a cell with a test substance and comparingclaudinyn-5 activity in the cell after exposure to the test substance tothat of an unexposed control cell. Preferably, claudinyn-5 activity isdetermined by measuring the permeability of the TJs using methods asmentioned above.

The present invention also provides animal models generated bymodulating the expression of the present protein in one or more tissuesof the animal. Such animals are useful for a number of purposes, becausethey represent an in vivo assay method for testing candidate moleculespotentially useful for the treatment of various pathophysiologicalaspects of diseases specifically related to the function of claudinyn-5.Study of the phenotype of such models can also allow the identificationof additional human equivalent diseases caused by or linked withclaudinyn-5 deficiency. These animals can be generated with any methodof targeting overexpression or inactivation of claudinyn-5. Such modelsare extremely useful, e.g. in the assessment of candidate therapies anddrugs for the treatment of inflammatory diseases and conditions.

In other embodiment, the current invention is used to diagnose diseasesor disorders associated with altered expression of claudinyn-5. Inparticular, it is useful in diagnosing patients with deficient amountsof claudinyn-5 which results in uncontrolled cell permeability. Examplesof such diseases and disorders include, but are not limited to, Crohn'sdisease, ulcerative colitis, irritable bowel syndrome, allergic asthma,gliobastoma, colorectal carcinoma, and adenomatous polyps. The methodincludes the steps of i) contacting a biological sample with a compoundcapable of selectively binding claudinyn-5 polypeptide or nucleic acids,and ii) detecting the level, or any other detectable property ofclaudinyn-5 in the sample. Preferably, a difference in the level orother property in the sample relative to in a control sample indicatesthe presence of the disease or disorder, or of a propensity fordeveloping the disease or disorder. Preferably, the biological sample isobtained from an individual suspected of suffering from the disease orcondition, or at risk of developing the disease or condition.Preferably, the biological sample comprises an epithelial cell sample. Apolyclonal or monoclonal antibody or any immunologically active fragmentspecific for claudinyn-5 may be used to detect levels of the protein.Preferably, the antibody or fragment thereof is detectably labeled with,for example, a fluorescent, radioactive, or otherwise detectablecompound. Detection can be carried out directly or indirectly with knownimmunohistological and immunofluorescing processes. For this, thereagents contained in the kit according to the invention can be directlylabeled with generally known molecules, including, but not limited to,enzymes such as alkaline phosphatase and peroxidase and fluorescent dyessuch as FITC, rhodamine, and Texas-Red. However, labeling can also occurindirectly by using secondary antibodies labeled with molecules such asbiotin, digoxigenin or the like and are then detected with a secondaryreagent. A method using a nucleic acid probe corresponding toclaudinyn-5 can also be used to determine the expression of claudinyn-5using, for example, well known PCR or RT-PCR techniques and inparticular in with “real-time” PCR system.

Alternatively, the present invention provides a tool to correlatemodulations in the expression of claudinyn-5 with certain pathologies.Thus, the present invention provides a novel candidate gene for suchconditions. Also in this embodiment, the determination of the level ofthe expression of claudinyn-5 within a biological sample provides anassay for an in vivo marker to classify and/or characterize of diseasestates. Preferably, such a method is applied to diagnose or classifypathologies such as Crohn's disease, ulcerative colitis, irritable bowelsyndrome, allergic asthma, gliobastoma, colorectal carcinoma, andadenomatous polyps. The invention thus includes test kits useful for thequantification of the amount of claudinyn-5 in a biological sample. Thekits comprise at least one immunological binding partner, e.g. amonoclonal or polyclonal antibody specific for claudinyn-5 and coupledto detectable markers. Application of such assays can be used to monitorthe progress of therapy administered to treat these or other conditions.Thus the assays may be applied in any situation wherein the level ofclaudinyn-5 expression can be used as an index of the condition,treatment, or effect of substances directly administered to the subjector to which the subject is exposed in the environment. Thus, thecondition of a patient can be monitored continuously and the quantifiedamount of claudinyn-5 polypeptides measured in the pathological samplecan be compared with the amount quantified in a biological sample of anormal individual or with the previous analysis of the same patient. Inthis embodiment, this marker can be measured by any suitable method,including immunoassays. It can also preferably be measured in tissuesand fluids recovered from inflammatory sites.

A further embodiment of the present invention is to provide novelmethods and compositions useful for the treatment of diseases andconditions associated with impaired TJ function. More preferably,claudinyn-5 polypeptide or fragment thereof can be used to treatdiseases associated with an impaired claudinyn-5 related function,including but not limited to, Crohn's disease, ulcerative colitis,irritable bowel syndrome, allergic asthma, gliobastoma, colorectalcarcinoma, and adenomatous polyps. Such methods comprise theadministration of a therapeutically-effective amount of claudinyn-5 tomammals suffering from the disease or condition, where “effectiveamount” is meant a concentration of claudinyn-5 capable of significantlyrestore a physiological TJs function. The compositions may beadministrated using any of the methods known in the art or discussedherein for delivering claudinyn-5 polynucleotides to a cell. Anyagonists of the expression or the activity of claudinyn-5 polypeptidesidentified as mentioned above can also be used in this embodiment. Suchcompositions are preferably delivered to an individual in combinationwith a physiologically acceptable carrier, such as a saline solution orother physiologically buffer suitable for administration to a patient.The particular amount of the compositions of the invention that will beadministered to the mammal for any particular condition will depend onthe clinical condition of the patient, and other factors such as theweight, age, and route of delivery. These compositions can optionallycomprise, one or more other compound of interest. This co-administrationmay be by simultaneous administration or by separate or sequentialadministrations. All of these additional components may be eitherobtained from natural sources or produced by recombinant geneticengineering techniques and/or chemical modification.

In a further embodiment, the present invention provides a method ofproducing “bioartificial” epithelia from non-epithelial cells. The“bioartificial” epithelia produced according to the invention providesvarious clinical applications for the generation of epithelia unable torepair or regenerate themselves. It can be used, for example, forreconstructive surgical procedures, for treating of disorders related toepithelial loss (for hereditary, traumatic or oncological reasons) orfor another therapeutic purposes (for example, burn treatments). Furtherpreferred is a method of using the nucleic acid corresponding toclaudinyn-5 wherein “bioartificial” epithelial cells are obtained bytransfection of said nucleic acid, and consequential remodeling, of thepatient's autologous cells not affected by any of the above conditions.The use of autologous cells in the preparation of the “bioartificial”epithelial cells of the invention in methods of treating disorders,conditions, or diseases associated with the loss of epithelial cellsreduces or eliminates the risk of tissue rejection typically observed intransplantation methodologies. Methods of bioartificial tissueengineering are generally known to those skilled in the art (Machens H.G. et al., Cells Tissues Organs 167: 88-94 (2000), the disclosure ofwhich is hereby incorporated by reference in its entirety).

Protein of SEQ ID NO:18 (internal Designation Clone 645730181-16-1-0-G9-F)

The cDNA of Clone 645730_(—)181-16-1-0-G9-F (SEQ ID NO:17) encodesBenzodiazepine Receptor 3 (BZRP-R3) protein of SEQ ID NO:18, comprisingthe amino acid sequence:MRLQGAIFVLLPHLGPILVWLFTRDHMSGWCEGPRMLSWCPFYKVLLLVQTAIYSVVGYASYLVWKDLGGGLGWPLALPLGLYADQLTISWTVLVLFFTVHN PGLALLHLLLLYGLVVSTALIWHPINKLAALLLLPYLAWLTVTSALTYHLWRDSLCPVHQPQPTEKSD. Accordingly, it willbe appreciated that all characteristics and uses of the polypeptides ofSEQ ID NO:18 described throughout the present application also pertainto the polypeptides encoded by the human cDNA included in Clone645730_(—)181-16-1-0-G9-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:17described throughout the present application also pertain to the nucleicacids comprising the human cDNA in Clone 645730_(—)181-16-1-0-G9-F. Apreferred embodiment of the invention is directed toward thecompositions of SEQ ID NO:17, SEQ ID NO:18 and Clone645730_(—)181-16-1-0-G9-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

The protein of SEQ ID NO:18, BZRP-R3, is a novel polymorphic variant ofhuman peripheral benzodiazepine receptor/isoquinoline binding protein(PBR/IBP) (accession numbers Q9Y531). BZRP-R3 displays fivetransmembrane-spanning segments: LQGAIFVLLPHLGPILVWLFT (SEQ ID NO:67),VLLLVQTAIYSVVGYASYLVW (SEQ ID NO:68), GLYADQLTISWTVLVLFFTVH (SEQ IDNO:69), GLALLHLLLLYGLVVSTALIW (SEQ ID NO:70), and LAALLLLPYLAWLTVTSALTY(SEQ ID NO:71). Accordingly, some embodiments of the present inventionrelate to polypeptides comprising the transmembrane domains. Moreover,BZRP-R3 displays a stretch of 11 amino acids (VTSALTYHLWR) (SEQ IDNO:72) that bind cholesterol. Accordingly, a preferred embodiment of thepresent invention comprises the amino acids of the cholesterolrecognition/interaction domain and the polynucleotides encoding thesame.

BZRP-R3 is capable of binding benzodiazepine and imidazopyridinederivatives, but is distinct from the GABA neurotransmitter receptor.BZRP-R3 polypeptides are most abundant in steroidogenic cells and arefound primarily on outer mitochondrial membranes. BZRP-R3 is associatedwith a 34-kDa pore-forming, voltage-dependent anion channel proteinlocated on the outer/inner mitochondrial membrane contact sites. Ligandsof BZRP-R3, upon binding to the receptor, simulate steroid synthesis insteroidogenic cells in vitro and in vivo. BZRP-R3 stimulates steroidformation by increasing the rate of cholesterol transfer from the outerto the inner mitochondrial membrane.

In addition to its role in mediating cholesterol movement acrossmembranes, BZRP-R3 has been implicated in several other physiologicalfunctions, including cell growth and differentiation, chemotaxis,mitochondrial physiology, porphyrin and heme biosynthesis, immuneresponse, and anion transport. In addition, BZRP-R3 agonists are potentanti-apoptotic compounds.

BZRP-R3 is associated with stress and anxiety disorders. BZRP-R3 plays arole in the regulation of several stress systems such as the HPA axis,the sympathetic nervous system, the renin-angiotensin axis, and theneuroendocrine axis. In these systems, acute stress typically leads toincreases in BZRP-R3 density, whereas chronic stress typically leads todecreases in BZRP-R3 density. For example, in Generalized AnxietyDisorder (GAD), Panic Disorder (PD), Generalized Social Phobia (GSP),and Post-Traumatic Stress Disorders (PTSD), BZRP-R3 density is typicallydecreased. BZRP-R3 is expressed glial cells in the brain. Furthermore,BZRP-R3 expression is increased in neurodegenerative disorders and afterneurotoxic and traumatic-ischemic brain damage. BZRP-R3 expression isdecreased in chronic schizophrenics, suggesting that the decreaseddensity of BZRP-R3 in the brain may be involved in the pathophysiologyof schizophrenia. However, BZRP-R3 is higher than normal in autopsiedbrain tissue from PSE patients (Portal-Systemic Encephalopathypatients).

BZRP-R3 increases mitochondrial activity and prevents apoptosis and istherefore implicated in tumor cell proliferation. BZRP-R3 ispreferentially expressed in liver and breast cancers. Further, BZRP-R3is useful as a tool/marker for detection, diagnosis, prognosis andtreatment of cancer.

Many ligands have been described that bind to BZRP-R3 with variousaffinities. Some benzodiazepines, Ro 5-4864 [4-chlorodiazepam], diazepamand structurally related compounds, are potent and selective PBRligands. Exogenous ligands also include 2-phenylquinoline carboxamides(PK11195 series), imidazo[1,2-a]pyridine-3-acetamides (Alpidem series),pyridazine, and isoquinilone derivatives. Some endogenous compounds,including porphyrins and diazepam binding inhibitor (DBI), bind toBZRP-R3.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a BZRP-R3 polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing BZRP-R3 expression.Preferably, the polynucleotides capable of directing BZRP-R3 expressionare located in the 5′ regulatory region of the BZRP-R3 gene. Furtherpreferably, these polynucleotides are located within 500 base pairs ofthe BZRP-R3 coding region. These polynucleotides preferably comprise apromoter sequence. Techniques known in the art for introducingpolynucleotide sequences to endogenous sequences are described in U.S.Pat. No. 5,641,670 and PCT WO9629411, which disclosures are herebyincorporated by reference in their entireties. BZRP-R3 protein producedby said host cell may be used for in vitro detection and purificationmethods as well as diagnosis and in vivo applications.

A preferred embodiment of the invention relates to compositions andmethods using the protein of the invention or fragment thereof to labelmitochondria in order to visualize any change in number, topology ormorphology of this organelle, for example in association with amitochondria-related human disorder, such as hereditary spasticparaplegia, neuroleptic malignant syndrome (NMS), the Rett syndrome,Alpers disease, or mitochondrial encephalomyopathies. For example, theprotein may be rendered easily detectable by inserting the cDNA encodingthe protein of the invention into a eukaryotic expression vector inframe with a sequence encoding a tag sequence. Eukaryotic cellsexpressing the tagged protein of the invention may also be used for thein vitro screening of drugs or genes capable of treating anymitochondria-related disease or conditions.

A preferred embodiment of the invention is a method to detectmitochondria comprising the step of contacting an antibody specific forBZRP-R3 with a cell. Preferably, the cell is fixed or otherwisepermeable to antibodies. Preferably, the antibody is labeled with anydetectable moiety including, but are not limited to, a fluorescentlabel, a radioactive atom, a paramagnetic ion, biotin, achemiluminescent label or a label which can be detected through asecondary enzymatic or binding step. The invention further provides amethod of diagnosing mitochondria-related disorders or conditions, suchas those listed above, and distinguishing these from other disorders.

In another embodiment, the protein of the invention may be used totarget heterologous compounds (polynucleotides) to steroidogenic cellsand/or the mitochondria. Such recombinant cDNA may be introduced, forexample, using in any vector, viral or non-viral, and viral vectors canbe but not limited to retroviral, adenoviral, and adeno-associatedvectors. For example, these heterologous polynucleotides may be used todeliver nucleic acids for mitochondrial gene therapy, i.e. to replace adefective mitochondrial gene and/or to inhibit the deleteriousexpression of a mitochondrial gene.

Another preferred embodiment of the invention is a method of screeningfor compounds that modulate the expression of BZRP-R3. This methodcomprises the steps of i) contacting a cell with a test compound and ii)comparing the level of BZRP-R3 polypeptides in a cell after exposure tothe test compound to that of an untreated control cell. The level ofBZRP-R3 polypeptides may be inferred by detecting mRNA for BZRP-R3 bymethods common to the art such as Northern blotting or RT-PCR. The levelof BZRP-R3 polypeptides may also be detected by antibody-based methodscommon to the art such as Western blotting or immunofluorescence. Testcompounds that increase BZRP-R3 expression are useful as agonists, asdiscussed herein. Test compounds that decrease BZRP-R3 expression areuseful as antagonists, as discussed herein.

Antagonists of BZRP-R3 include agents which decrease the levels ofexpressed mRNA encoding the protein of SEQ ID NO:18. These include, butare not limited to, RNAi, one or more ribozymes capable of digesting theprotein of the invention, or antisense oligonucleotides capable ofhybridizing to mRNA encoding BZRP-R3. Antisense oligonucleotides can beadministrated as DNA, RNA, as DNA entrapped in proteoliposomescontaining viral envelope receptor proteins [Kanoda, Y. et al. (1989)Science 243: 375, which disclosure is hereby incorporated by referencein its entirety] or as part of a vector which can be expressed in thetarget cell to provide antisense DNA or RNA. Vectors which are expressedin particular cell types are known in the art. Alternatively, the DNAcan be injected along with a carrier. A carrier can be a protein such asa cytokine, for example interleukin 2, or polylysine-glycoproteincarriers. Carrier proteins, vectors, and methods of making and usingpolylysine carrier systems are known in the art. Alternatively, nucleicacids encoding antisense molecules may be coated onto gold beads andintroduced into the skin with, for example, a gene gun [Ulmer, J. B. etal. (1993) Science 259:1745, which disclosure is hereby incorporated byreference in its entirety].

A preferred embodiment of the invention is a method of screening forcompounds that bind to BZRP-R3 polypeptides. Such compounds are usefulfor developing agonists and antagonists of BZRP-R3 activity. This methodcomprises the steps of: i) contacting a BZRP-R3 polypeptide or fragmentthereof with a test compound under conditions that allow binding tooccur and ii) detecting binding of said test compound. Binding may bedetected by any method common to the art such as competition with alabeled antibody specific for BZRP-R3 or by direct labeling of each testsubstance. In one example of such a method, a polynucleotide encoding aBZRP-R3 polypeptide or a biologically active fragment thereof istransformed into a eukaryotic or prokaryotic host cell. The transformedcells may be viable or fixed. Drugs or compounds which are candidatesfor binding BZRP-R3 polypeptides are screened against such transformedcells in binding assays well known to those skilled in the art.Alternatively, assays such as those taught in Geysen H. N., WOApplication 84/03564, published on Sep. 13, 1984, and incorporatedherein by reference in its entirety, may be used to screen for peptidecompounds which demonstrate binding affinity for BZRP-R3 polypeptides orfragments thereof. In another embodiment, competitive drug screeningassays using neutralizing antibodies specifically compete with a testcompound for binding to BZRP-R3 polypeptides or fragments thereof.Preferred test compounds are those included in the benzodiazepine class,such as diazepam (i.e., valium), triazolobenzodiazepine, and adinazolam,as well as modified versions thereof. Further preferred test compoundsare in the imidazopyridine and isoquinilone classes. Each test compoundmay additionally be excluded individually or as a class.

A variety of drug screening techniques may be employed. In this aspectof the invention, BZRP-R3 polypeptide or fragments thereof, may be freein solution, affixed to a solid support, recombinantly expressed on orchemically attached to a cell surface, or located intracellularly. Theformation of binding complexes between BZRP-R3 polypeptides or fragmentsthereof, and the compound being tested, may then be measured asdescribed.

Another embodiment of the subject invention provides compositions andmethods of selectively modulating the activity of the protein of theinvention. Modulation of BZRP-R3 allows for the successful prevention,treatment, or management of disorders or biochemical abnormalitiesassociated with BZRP-R3. Agonist compounds are those that increase theamount of BZRP-R3 polypeptides in a cell or increase the biologicalactivity of BZRP-R3. A preferred embodiment of the invention is a methodof screening for agonists that bind to BZRP-R3 comprising the steps of:i) screening for test substances that bind to BZRP-R3, as describedabove and ii) detecting BZRP-R3 biological activity. Preferably, thismethod is accomplished in an intact cell. Further preferably, the cellis a steroidogenic cell such as a testicular or ovarian cell.Preferably, the biological activity of BZRP-R3 is determined bymeasuring the concentration of steroid hormones released from the cellbefore and after exposure to the test substance. Agonists of BZRP-R3will increase the release of steroid hormones from the cell. Antagonistcompounds are those that decrease the amount of BZRP-R3 polypeptides ina cell or decrease the biological activity of BZRP-R3. Another preferredembodiment of the invention is a method of screening for antagoniststhat bind to BZRP-R3 comprising the steps of: i) screening for testsubstances that bind to BZRP-R3, as described above and ii) detectingBZRP-R3 biological activity. Preferably, this method is accomplished inan intact cell. Further preferably, the cell is a steroidogenic cellsuch as a testicular or ovarian cell. Preferably, the biologicalactivity of BZRP-R3 is determined by measuring the concentration ofsteroid hormones released from the cell before and after exposure to thetest substance. Antagonists of BZRP-R3 will decrease the release ofsteroid hormones from the cell.

Antagonists, able to reduce or inhibit the expression or the activity ofthe protein of the invention, are useful in the treatment of diseasesassociated with elevated levels of BZRP-R3, increased cell proliferationor reduced apoptosis, and increased cholesterol transport. Thus, thesubject invention provides methods for treating a variety of diseases ordisorders, including but not limited to cancers, especially liver andbreast cancer, and portal-systemic encephalopathy. Increased cholesteroltransport into the mitochondria of steroidogenic cells results in higherthan normal production of steroid hormones such as progesterone,testosterone, and estrogen. Abnormally high levels of steroid hormoneslead to disruption of adrenocortical feedback mechanisms andunderproduction of trophic hormones from the hypothalamus and pituitary.Inhibition of BZRP-R3 and steroidogenesis may increase levels of trophichormones such as gonadotropin-releasing hormone.

Alternatively, the subject invention provides a method of treatingdiseases or disorders associated with decreased levels of BZRP-R3polypeptides and decreased steroid hormone release with an agonistthereof. Such method comprises the step of contacting a cell with aBZRP-R3 agonist. This method comprises the step of contacting a cellwith an agonist of BZRP-R3. Thus, the subject invention provides methodsof treating disorders including, but not limited to, schizophrenia,chronic stress, GAD, PD, GSP and PTSD. Other disorders which may betreated by agonists of BZRP-R3 include those associated with decreasesin cell proliferation, e.g. developmental retardation. Furthermore,because BZRP-R3 is able to transport cholesterol into cells, BZRP-R3agonists may also be used to increase cholesterol transport into cells.Diseases associated with cholesterol transport deficiencies includelipoidal adrenal hyperplasia, ovarian cysts, abnormal lipid deposits insteroidogenic cells. Disorders that reflect a requirement forcholesterol for myelin and myelination, include Alzheimer's disease,multiple sclerosis, spinal cord injury, and brain developmentneuropathy. The methods of treating disorders associated with decreasedlevels of BZRP-R3 may be practiced by introducing agonists whichstimulate the expression or the activity of BZRP-R3.

Additionally, disorders resulting from defective mitochondrial activitymay be treated with an agonist to BZRP-R3. Defective mitochondrialactivity may alternatively or additionally result in the generation ofhighly reactive free radicals that have the potential of damaging cellsand tissues. These free radicals may include reactive oxygen species(ROS) such as superoxide, peroxynitrite and hydroxyl radicals, and otherreactive species that may be toxic to cells and cause apoptosis. Forexample, oxygen free radical induced lipid peroxidation is awell-established pathogenic mechanism in central nervous system (CNS)injury such as that found in a number of degenerative diseases, and inischemia (i.e., stroke). Diseases associated with altered mitochondrialfunction and apoptosis include: Alzheimer's Disease, diabetes mellitus,Parkinson's Disease, Huntington's disease, dystonia, Leber's hereditaryoptic neuropathy, schizophrenia, mitochondrial encephalopathy, lacticacidosis, and stroke.

A further preferred embodiment includes a method of inhibiting apoptosisof cells in culture. This method comprises the step of contacting a cellin culture with an agonist to BZRP-R3. Such methods are useful forculturing cells that are notoriously undergo apoptosis, such as primaryneurons and lymphocytes.

In one embodiment, the level of BZRP-R3 in a cell may be increased byintroducing nucleic acids encoding a BZRP-R3 polypeptide or biologicallyactive fragment thereof into a targeted cell type. Vectors useful insuch methods are known to those skilled in the art, as are methods ofintroducing such nucleic acids into target tissues.

Antibodies or other polypeptides capable of reducing or inhibiting theactivity of BZRP-R3 may be provided as in isolated and substantiallypurified form. Alternatively, antibodies or other polypeptides capableof inhibiting or reducing the activity of BZRP-R3 may be recombinantlyexpressed in the target cell to provide a modulating effect. Inaddition, compounds which inhibit or reduce the activity of BZRP-R3 maybe incorporated into biodegradable polymers being implanted in thevicinity of where drug delivery is desired. For example, biodegradablepolymers may be implanted at the site of a tumor or, alternatively,biodegradable polymers containing antagonists/agonists may be implantedto slowly release the compounds systemically. Biodegradable polymers,and their use, are known to those of skill in the art (see, for example,Brem et al. (1991) J. Neurosurg. 74:441-446, which disclosure is herebyincorporated by reference in its entirety).

In another embodiment, the invention provides methods and compositionsfor detecting the level of expression of the mRNA encoding the proteinof the invention. Quantification of mRNA levels of BZRP-R3 may be usefulfor the diagnosis or prognosis of diseases associated with an alteredexpression of the protein of the invention. Assays for the detection andquantification of the mRNA encoding BZRP-R3 are well known in the art(see, for example, Maniatis, Fitsch and Sambrook, Molecular Cloning; ALaboratory Manual (1982), or Current Protocols in Molecular Biology,Ausubel, F. M. et al. (Eds), Wiley & Sons, Inc., disclosures of whichare hereby incorporated by reference in their entireties).

Polynucleotides probes or primers for the detection of BZRP-R3 mRNA canbe designed from the cDNA of SEQ ID NO:17. Methods for designing probesand primers are known in the art. In another embodiment, the subjectinvention provides diagnostic kits for the detection of the mRNA of theprotein of the invention in cells. The kit comprises a package havingone or more containers of oligonucleotide primers for detection of thepolynucleotides of the invention in PCR assays or one or more containersof polynucleotide probes for the detection of the BZRP-R3 mRNA by insitu hybridization or Northern analysis. Kits may, optionally, includecontainers of various reagents used in various hybridization assays. Thekit may also, optionally, contain one or more of the following items:polymerization enzymes, buffers, instructions, controls, or detectionlabels. Kits may also, optionally, include containers of reagents mixedtogether in suitable proportions for performing the hybridization assaymethods in accordance with the invention. Reagent containers preferablycontain reagents in unit quantities that obviate measuring steps whenperforming the subject methods.

In another embodiment, the invention relates to methods and compositionsfor detecting and quantifying the level of the protein of the inventionpresent in a particular biological sample. These methods are useful forthe diagnosis or prognosis of diseases associated with altered levels ofthe protein of the invention. Diagnostic assays to detect the protein ofthe invention may comprise a biopsy, in situ assay of cells from organor tissue sections, or an aspirate of cells from a tumor or normaltissue. In addition, assays may be conducted upon cellular extracts fromorgans, tissues, cells, urine, or serum or blood or any other body fluidor extract.

Assays for the quantification of BZRP-R3 polypeptides may be performedaccording to methods well known in the art. Typically, these assayscomprise the steps of: contacting the sample with a ligand of theprotein of the invention or an antibody (polyclonal or monoclonal) thatspecifically recognizes the protein of the invention or a fragmentthereof and detecting the complex formed between the protein of theinvention present in the sample and the ligand or antibody.

Fragments of the ligands and antibodies may also be used in the bindingassays, provided these fragments are capable of specifically interactingwith BZRP-R3 polypeptides. Further, ligands and antibodies which bind toBZRP-R3 may be labeled according to methods known in the alt. Labelswhich are useful in the subject invention include, but are not limitedto, enzymes labels, radioisotopic labels, paramagnetic labels, andchemiluminescent labels. Common techniques are described by Kennedy, J.H., et al. (1976) Clin. Chim. Acta 70:1-31; and Schurs, A. H. et al.(1977) Clin. Chim. Acta 81: 1-40, disclosures of which are herebyincorporated by reference in their entireties.

The subject invention also provides methods and compositions for theidentification of metastatic tumor masses. In this aspect of theinvention, the polypeptide or antibody that specifically binds a BZRP-R3polypeptide or fragment thereof may be used as a marker for theidentification of the metastatic tumor mass. Metastatic tumorsoriginating from the breast or liver may overexpress BZRP-R3polypeptides, whereas newly forming tumors, or those originating fromother tissues are not expected to bear BZRP-R3.

Protein of SEQ ID NO:20 (Internal Designation Clone 646762181-21-2-0-A3-F)

The cDNA of Clone 646762_(—)181-21-2-0-A3-F (SEQ ID NO:19) encodesBenzodiazepine Receptor 4 (BZRP-R4) protein of SEQ ID NO:20, comprisingthe amino acid sequence:MRLQGAIFVLLPHLGPILVWLFTRDHMSGLCEGPRMLSWCPFYKVLLLVQTAIYSVVGYASYLVWKDLGGGLGWPLALPLGLYAVQLTISWTVLVLFFTVHNPGLALLHLLLLYGLVVSTALIWHPINKLAALLLLPYLAWLTVTSALTYHLWRDSLCPVHQPQPTEKSD. Accordingly, it willbe appreciated that all characteristics and uses of the polypeptides ofSEQ ID NO:20 described throughout the present application also pertainto the polypeptides encoded by the human cDNA included in Clone646762_(—)181-21-2-0-A3-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:19described throughout the present application also pertain to the nucleicacids comprising the human cDNA in Clone 646762_(—)181-21-2-0-A3-F. Apreferred embodiment of the invention is directed toward thecompositions of SEQ ID NO:19, SEQ ID NO:20 and Clone646762_(—)181-21-2-0-A3-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

The protein of SEQ ID NO:20, BZRP-R4, is a novel polymorphic variant ofhuman peripheral benzodiazepine receptor/isoquinoline binding protein(PBR/IBP) (accession numbers Q9Y531). BZRP-R4 displays fivetransmembrane-spanning segments: LQGAIFVLLPHLGPILVWLFT (SEQ ID NO: 67),VLLLVQTAIYSVVGYASYLVW (SEQ ID NO:68), LYAVQLTISWTVLVLFFTVHN (SEQ IDNO:73), LALLHLLLLYGLVVSTALIWH (SEQ ID NO:74), and LAALLLLPYLAWLTVTSALTY(SEQ ID NO:71). Accordingly, some embodiments of the present inventionrelate to polypeptides comprising the transmembrane domains. Moreover,BZRP-R4 displays a stretch of 11 amino acids (VTSALTYHLWR) (SEQ IDNO:72) that bind cholesterol. Accordingly, a preferred embodiment of thepresent invention comprises the amino acids of the cholesterolrecognition/interaction domain and the polynucleotides encoding thesame.

BZRP-R4 is capable of binding benzodiazepine and imidazopyridinederivatives, but is distinct from the GABA neurotransmitter receptor.BZRP-R4 polypeptides are most abundant in steroidogenic cells and arefound primarily on outer mitochondrial membranes. BZRP-R4 is associatedwith a 34-kDa pore-forming, voltage-dependent anion channel proteinlocated on the outer/inner mitochondrial membrane contact sites. Ligandsof BZRP-R4, upon binding to the receptor, simulate steroid synthesis insteroidogenic cells in vitro and in vivo. BZRP-R4 stimulates steroidformation by increasing the rate of cholesterol transfer from the outerto the inner mitochondrial membrane.

In addition to its role in mediating cholesterol movement acrossmembranes, BZRP-R4 has been implicated in several other physiologicalfunctions, including cell growth and differentiation, chemotaxis,mitochondrial physiology, porphyrin and heme biosynthesis, immuneresponse, and anion transport. In addition, BZRP-R4 agonists are potentanti-apoptotic compounds.

BZRP-R4 is associated with stress and anxiety disorders. BZRP-R4 plays arole in the regulation of several stress systems such as the HPA axis,the sympathetic nervous system, the renin-angiotensin axis, and theneuroendocrine axis. In these systems, acute stress typically leads toincreases in BZRP-R4 density, whereas chronic stress typically leads todecreases in BZRP-R4 density. For example, in Generalized AnxietyDisorder (GAD), Panic Disorder (PD), Generalized Social Phobia (GSP),and Post-Traumatic Stress Disorders (PTSD), BZRP-R4 density is typicallydecreased. BZRP-R4 is expressed glial cells in the brain. Furthermore,BZRP-R4 expression is increased in neurodegenerative disorders and afterneurotoxic and traumatic-ischemic brain damage. BZRP-R4 expression isdecreased in chronic schizophrenics, suggesting that the decreaseddensity of BZRP-R4 in the brain may be involved in the pathophysiologyof schizophrenia. However, BZRP-R4 is higher than normal in autopsiedbrain tissue from PSE patients (Portal-Systemic Encephalopathypatients).

BZRP-R4 increases mitochondrial activity and prevents apoptosis and istherefore implicated in tumor cell proliferation. BZRP-R4 ispreferentially expressed in liver and breast cancers. Further, BZRP-R4is useful as a tool/marker for detection, diagnosis, prognosis andtreatment of cancer.

Many ligands have been described that bind to BZRP-R4 with variousaffinities. Some benzodiazepines, Ro 5-4864 [4-chlorodiazepam], diazepamand structurally related compounds, are potent and selective PBRligands. Exogenous ligands also include 2-phenylquinoline carboxamides(PK11195 series), imidazo[1,2-a]pyridine-3-acetamides (Alpidem series),pyridazine, and isoquinilone derivatives. Some endogenous compounds,including porphyrins and diazepam binding inhibitor (DBI), bind toBZRP-R4.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a BZRP-R4 polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing BZRP-R4 expression.Preferably, the polynucleotides capable of directing BZRP-R4 expressionare located in the 5′ regulatory region of the BZRP-R4 gene. Furtherpreferably, these polynucleotides are located within 500 base pairs ofthe BZRP-R4 coding region. These polynucleotides preferably comprise apromoter sequence. Techniques known in the art for introducingpolynucleotide sequences to endogenous sequences are described in U.S.Pat. No. 5,641,670 and PCT WO9629411, which disclosures are herebyincorporated by reference in their entireties. BZRP-R4 protein producedby said host cell may be used for in vitro detection and purificationmethods as well as diagnosis and in vivo applications.

A preferred embodiment of the invention relates to compositions andmethods using the protein of the invention or fragment thereof to labelmitochondria in order to visualize any change in number, topology ormorphology of this organelle, for example in association with amitochondria-related human disorder, such as hereditary spasticparaplegia, neuroleptic malignant syndrome (NMS), the Rett syndrome,Alpers disease, or mitochondrial encephalomyopathies. For example, theprotein may be rendered easily detectable by inserting the cDNA encodingthe protein of the invention into a eukaryotic expression vector inframe with a sequence encoding a tag sequence. Eukaryotic cellsexpressing the tagged protein of the invention may also be used for thein vitro screening of drugs or genes capable of treating anymitochondria-related disease or conditions.

A preferred embodiment of the invention is a method to detectmitochondria comprising the step of contacting an antibody specific forBZRP-R4 with a cell. Preferably, the cell is fixed or otherwisepermeable to antibodies. Preferably, the antibody is labeled with anydetectable moiety including, but are not limited to, a fluorescentlabel, a radioactive atom, a paramagnetic ion, biotin, achemiluminescent label or a label which can be detected through asecondary enzymatic or binding step. The invention further provides amethod of diagnosing mitochondria-related disease or conditions, forexample, but not limited to, those listed above, and distinguishing suchdiseases from other diseases.

In another embodiment, the protein of the invention may be used totarget heterologous compounds (polynucleotides) to steroidogenic cellsand/or the mitochondria. Such recombinant cDNA may be introduced, forexample, using in any vector, viral or non-viral, and viral vectors canbe but not limited to retroviral, adenoviral, and adeno-associatedvectors. For example, these heterologous polynucleotides may be used todeliver nucleic acids for mitochondrial gene therapy, i.e. to replace adefective mitochondrial gene and/or to inhibit the deleteriousexpression of a mitochondrial gene.

Another preferred embodiment of the invention is a method of screeningfor compounds that modulate the expression of BZRP-R4. This methodcomprises the steps of i) contacting a cell with a test compound and ii)comparing the level of BZRP-R4 polypeptides in a cell after exposure tothe test compound to that of an untreated control cell. The level ofBZRP-R4 polypeptides may be inferred by detecting mRNA for BZRP-R4 bymethods common to the art such as Northern blotting or RT-PCR. The levelof BZRP-R4 polypeptides may also be detected by antibody-based methodscommon to the art such as Western blotting or immunofluorescence. Testcompounds that increase BZRP-R4 expression are useful as agonists, asdiscussed herein. Test compounds that decrease BZRP-R4 expression areuseful as antagonists, as discussed herein.

Antagonists of BZRP-R4 include agents which decrease the levels ofexpressed mRNA encoding the protein of SEQ ID NO:20. These include, butare not limited to, RNAi, one or more ribozymes capable of digesting theprotein of the invention, or antisense oligonucleotides capable ofhybridizing to mRNA encoding BZRP-R4. Antisense oligonucleotides can beadministrated as DNA, RNA, as DNA entrapped in proteoliposomescontaining viral envelope receptor proteins [Kanoda, Y. et al. (1989)Science 243: 375, which disclosure is hereby incorporated by referencein its entirety] or as part of a vector which can be expressed in thetarget cell to provide antisense DNA or RNA. Vectors which are expressedin particular cell types are known in the art. Alternatively, the DNAcan be injected along with a carrier. A carrier can be a protein such asa cytokine, for example interleukin 2, or polylysine-glycoproteincarriers. Carrier proteins, vectors, and methods of making and usingpolylysine carrier systems are known in the art. Alternatively, nucleicacids encoding antisense molecules may be coated onto gold beads andintroduced into the skin with, for example, a gene gun [Ulmer, J. B. etal. (1993) Science 259:1745, which disclosure is hereby incorporated byreference in its entirety].

A preferred embodiment of the invention is a method of screening forcompounds that bind to BZRP-R4 polypeptides. Such compounds are usefulfor developing agonists and antagonists of BZRP-R4 activity. This methodcomprises the steps of: i) contacting a BZRP-R4 polypeptide or fragmentthereof with a test compound under conditions that allow binding tooccur and ii) detecting binding of said test compound. Binding may bedetected by any method common to the art such as competition with alabeled antibody specific for BZRP-R4 or by direct labeling of each testsubstance. In one example of such a method, a polynucleotide encoding aBZRP-R4 polypeptide or a biologically active fragment thereof istransformed into a eukaryotic or prokaryotic host cell. The transformedcells may be viable or fixed. Drugs or compounds which are candidatesfor binding BZRP-R4 polypeptides are screened against such transformedcells in binding assays well known to those skilled in the art.Alternatively, assays such as those taught in Geysen H. N., WOApplication 84/03564, published on Sep. 13, 1984, and incorporatedherein by reference in its entirety, may be used to screen for peptidecompounds which demonstrate binding affinity for BZRP-R4 polypeptides orfragments thereof. In another embodiment, competitive drug screeningassays using neutralizing antibodies specifically compete with a testcompound for binding to BZRP-R4 polypeptides or fragments thereof.Preferred test compounds are those included in the benzodiazepine class,such as diazepam (i.e., valium), triazolobenzodiazepine, and adinazolam,as well as modified versions thereof. Further preferred test compoundsare in the imidazopyridine and isoquinilone classes. Each test compoundmay additionally be excluded individually or as a class.

A variety of drug screening techniques may be employed. In this aspectof the invention, BZRP-R4 polypeptide or fragments thereof, may be freein solution, affixed to a solid support, recombinantly expressed on orchemically attached to a cell surface, or located intracellularly. Theformation of binding complexes between BZRP-R4 polypeptides or fragmentsthereof, and the compound being tested, may then be measured asdescribed.

Another embodiment of the subject invention provides compositions andmethods of selectively modulating the activity of the protein of theinvention. Modulation of BZRP-R4 allows for the successful prevention,treatment, or management of disorders or biochemical abnormalitiesassociated with BZRP-R4. Agonist compounds are those that increase theamount of BZRP-R4 polypeptides in a cell or increase the biologicalactivity of BZRP-R4. A preferred embodiment of the invention is a methodof screening for agonists that bind to BZRP-R4 comprising the steps of:i) screening for test substances that bind to BZRP-R4, as describedabove and ii) detecting BZRP-R4 biological activity. Preferably, thismethod is accomplished in an intact cell. Further preferably, the cellis a steroidogenic cell such as a testicular or ovarian cell.Preferably, the biological activity of BZRP-R4 is determined bymeasuring the concentration of steroid hormones released from the cellbefore and after exposure to the test substance. Agonists of BZRP-R4will increase the release of steroid hormones from the cell. Antagonistcompounds are those that decrease the amount of BZRP-R4 polypeptides ina cell or decrease the biological activity of BZRP-R4. Another preferredembodiment of the invention is a method of screening for antagoniststhat bind to BZRP-R4 comprising the steps of: i) screening for testsubstances that bind to BZRP-R4, as described above and ii) detectingBZRP-R4 biological activity. Preferably, this method is accomplished inan intact cell. Further preferably, the cell is a steroidogenic cellsuch as a testicular or ovarian cell. Preferably, the biologicalactivity of BZRP-R4 is determined by measuring the concentration ofsteroid hormones released from the cell before and after exposure to thetest substance. Antagonists of BZRP-R4 will decrease the release ofsteroid hormones from the cell.

Antagonists, able to reduce or inhibit the expression or the activity ofthe protein of the invention, are useful in the treatment of diseasesassociated with elevated levels of BZRP-R4, increased cell proliferationor reduced apoptosis, and increased cholesterol transport. Thus, thesubject invention provides methods for treating a variety of diseases ordisorders, including but not limited to cancers, especially liver andbreast cancer, and portal-systemic encephalopathy. Increased cholesteroltransport into the mitochondria of steroidogenic cells results in higherthan normal production of steroid hormones such as progesterone,testosterone, and estrogen. Abnormally high levels of steroid hormoneslead to disruption of adrenocortical feedback mechanisms andunderproduction of trophic hormones from the hypothalamus and pituitary.Inhibition of BZRP-R4 and steroidogenesis may increase levels of trophichormones such as gonadotropin-releasing hormone.

Alternatively, the subject invention provides a method of treatingdiseases or disorders associated with decreased levels of BZRP-R4polypeptides and decreased steroid hormone release with an agonistthereof. Such method comprises the step of contacting a cell with aBZRP-R4 agonist. This method comprises the step of contacting a cellwith an agonist of BZRP-R4. Thus, the subject invention provides methodsof treating disorders including, but not limited to, schizophrenia,chronic stress, GAD, PD, GSP and PTSD. Other disorders which may betreated by agonists of BZRP-R4 include those associated with decreasesin cell proliferation, e.g. developmental retardation. Furthermore,because BZRP-R4 is able to transport cholesterol into cells, BZRP-R4agonists may also be used to increase cholesterol transport into cells.Diseases associated with cholesterol transport deficiencies includelipoidal adrenal hyperplasia, ovarian cysts, abnormal lipid deposits insteroidogenic cells. Disorders that reflect a requirement forcholesterol for myelin and myelination, include Alzheimer's disease,multiple sclerosis, spinal cord injury, and brain developmentneuropathy. The methods of treating disorders associated with decreasedlevels of BZRP-R4 may be practiced by introducing agonists whichstimulate the expression or the activity of BZRP-R4.

Additionally, disorders resulting from defective mitochondrial activitymay be treated with an agonist to BZRP-R4. Defective mitochondrialactivity may alternatively or additionally result in the generation ofhighly reactive free radicals that have the potential of damaging cellsand tissues. These free radicals may include reactive oxygen species(ROS) such as superoxide, peroxynitrite and hydroxyl radicals, and otherreactive species that may be toxic to cells and cause apoptosis. Forexample, oxygen free radical induced lipid peroxidation is awell-established pathogenic mechanism in central nervous system (CNS)injury such as that found in a number of degenerative diseases, and inischemia (i.e., stroke). Diseases associated with altered mitochondrialfunction and apoptosis include: Alzheimer's Disease, diabetes mellitus,Parkinson's Disease, Huntington's disease, dystonia, Leber's hereditaryoptic neuropathy, schizophrenia, mitochondrial encephalopathy, lacticacidosis, and stroke.

A further preferred embodiment includes a method of inhibiting apoptosisof cells in culture. This method comprises the step of contacting a cellin culture with an agonist to BZRP-R4. Such methods are useful forculturing cells that are notoriously undergo apoptosis, such as primaryneurons and lymphocytes.

In one embodiment, the level of BZRP-R4 in a cell may be increased byintroducing nucleic acids encoding a BZRP-R4 polypeptide or biologicallyactive fragment thereof into a targeted cell type. Vectors useful insuch methods are known to those skilled in the art, as are methods ofintroducing such nucleic acids into target tissues.

Antibodies or other polypeptides capable of reducing or inhibiting theactivity of BZRP-R4 may be provided as in isolated and substantiallypurified form. Alternatively, antibodies or other polypeptides capableof inhibiting or reducing the activity of BZRP-R4 may be recombinantlyexpressed in the target cell to provide a modulating effect. Inaddition, compounds which inhibit or reduce the activity of BZRP-R4 maybe incorporated into biodegradable polymers being implanted in thevicinity of where drug delivery is desired. For example, biodegradablepolymers may be implanted at the site of a tumor or, alternatively,biodegradable polymers containing antagonists/agonists may be implantedto slowly release the compounds systemically. Biodegradable polymers,and their use, are known to those of skill in the art (see, for example,Brem et al. (1991) J. Neurosurg. 74:441-446, which disclosure is herebyincorporated by reference in its entirety).

In another embodiment, the invention provides methods and compositionsfor detecting the level of expression of the mRNA encoding the proteinof the invention. Quantification of mRNA levels of BZRP-R4 may be usefulfor the diagnosis or prognosis of diseases associated with an alteredexpression of the protein of the invention. Assays for the detection andquantification of the mRNA encoding BZRP-R4 are well known in the art(see, for example, Maniatis, Fitsch and Sambrook, Molecular Cloning; ALaboratory Manual (1982), or Current Protocols in Molecular Biology,Ausubel, F. M. et al. (Eds), Wiley & Sons, Inc., disclosures of whichare hereby incorporated by reference in their entireties).

Polynucleotides probes or primers for the detection of BZRP-R4 mRNA canbe designed from the cDNA of SEQ ID NO:19. Methods for designing probesand primers are known in the art. In another embodiment, the subjectinvention provides diagnostic kits for the detection of the mRNA of theprotein of the invention in cells. The kit comprises a package havingone or more containers of oligonucleotide primers for detection of thepolynucleotides of the invention in PCR assays or one or more containersof polynucleotide probes for the detection of the BZRP-R4 mRNA by insitu hybridization or Northern analysis. Kits may, optionally, includecontainers of various reagents used in various hybridization assays. Thekit may also, optionally, contain one or more of the following items:polymerization enzymes, buffers, instructions, controls, or detectionlabels. Kits may also, optionally, include containers of reagents mixedtogether in suitable proportions for performing the hybridization assaymethods in accordance with the invention. Reagent containers preferablycontain reagents in unit quantities that obviate measuring steps whenperforming the subject methods.

In another embodiment, the invention relates to methods and compositionsfor detecting and quantifying the level of the protein of the inventionpresent in a particular biological sample. These methods are useful forthe diagnosis or prognosis of diseases associated with altered levels ofthe protein of the invention. Diagnostic assays to detect the protein ofthe invention may comprise a biopsy, in situ assay of cells from organor tissue sections, or an aspirate of cells from a tumor or normaltissue. In addition, assays may be conducted upon cellular extracts fromorgans, tissues, cells, urine, or serum or blood or any other body fluidor extract.

Assays for the quantification of BZRP-R4 polypeptides may be performedaccording to methods well known in the art. Typically, these assayscomprise the steps of: contacting the sample with a ligand of theprotein of the invention or an antibody (polyclonal or monoclonal) thatspecifically recognizes the protein of the invention or a fragmentthereof and detecting the complex formed between the protein of theinvention present in the sample and the ligand or antibody. Fragments ofthe ligands and antibodies may also be used in the binding assays,provided these fragments are capable of specifically interacting withBZRP-R4 polypeptides. Further, ligands and antibodies which bind toBZRP-R4 may be labeled according to methods known in the art. Labelswhich are useful in the subject invention include, but are not limitedto, enzymes labels, radioisotopic labels, paramagnetic labels, andchemiluminescent labels. Typical techniques are described by Kennedy, J.H., et al. (1976) Clin. Chim. Acta 70:1-31; and Schurs, A. H. et al.(1977) Clin. Chim. Acta 81: 1-40, disclosures of which are herebyincorporated by reference in their entireties.

The subject invention also provides methods and compositions for theidentification of metastatic tumor masses. In this aspect of theinvention, the polypeptide or antibody that specifically binds a BZRP-R4polypeptide or fragment thereof may be used as a marker for theidentification of the metastatic tumor mass. Metastatic tumors whichoriginated from the breast or liver may overexpress BZRP-R4polypeptides, whereas newly forming tumors, or those originating fromother tissues are not expected to bear BZRP-R4.

Protein of SEQ ID NO:22 (Internal Designation Clone 420594145-19-4-0-E7-F)

The cDNA of Clone 420594 145-19-4-0-E7-F (SEQ ID NO:21) encodes the 106amino acid Scolakin protein of SEQ ID NO:22 comprising the amino acidsequence: MPFLDIQKRFGLNIDRWLTIQSCEQPYKMAGRCHAFEKEWIECAHGIGYTRAEKECKIEYDDFVECLLRQKTMRRAGTIRKQRDKLIKEGKYTPPPHHIGKGEPWP. Accordingly, it will beappreciated that all characteristics and uses of polypeptides of SEQ IDNO:22 described throughout the present application also pertain to thepolypeptides encoded by the human cDNA included in Clone 420594145-19-4-0-E7-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:21described throughout the present application also pertain to the nucleicacids comprising the human cDNA in Clone 420594 145-19-4-0-E7-F. Alsopreferred are fragments having a biological activity as describedtherein and the polynucleotides encoding the fragments.

Scolakin, the protein of SEQ ID NO:22, represents a novel variant formof the 15 kilodalton NADH dehydrogenase subunit NDUFS5 (Genbank entryAF020352).

NADH dehydrogenase (NADH:ubiquinone oxidoreductase, complex I) is thefirst multisubunit inner membrane protein complex in a chain of threecomplexes that make up the mitochondrial electron transport chain. Themitochondrial electron transport chain is responsible for the transportof electrons from NADH to oxygen and the coupling of this oxidation tothe synthesis of ATP (oxidative phosphorylation) which provides theenergy source for driving a cell's many energy-requiring reactions. NADHdehydrogenase accomplishes the first step in this process by acceptingtwo electrons from NADH and passing them through a flavin molecule toubiquinone, which transfers the electrons to the second enzyme complexin the chain. It contains a number of prosthetic groups which areinvolved in the transfer of electrons, namely, flavin mononucleotide(FMN) and at least six iron-sulphur clusters (binuclear andtetranuclear). NADH dehydrogenase is the largest of the three complexeswith an estimated mass of 800 kDa comprising about 41 polypeptides ofwidely varying size and composition. Seven of these complex Ipolypeptides are encoded by mitochondrial DNA while the remaining arenuclear gene products that are imported into the mitochondria. Thepolypeptide composition of NADH dehydrogenase is very similar in avariety of mammalian species including rat, rabbit, cow, and man. Inman, deficiency of NADH dehydrogenase is one of the most frequent causesof human encephalomyopathies. Defects and altered expression of thisenzyme have also been reported to be associated with other diseaseconditions, i.e., neurodegenerative diseases and cancer. In addition,NADH dehydrogenase reduction of the quinone moiety in chemotherapeuticagents such as doxorubicin is believed to contribute to the antitumoractivity and/or mutagenicity of these drugs. Defects inmitochondrial-encoded subunits of complex I have been described. Howevera relatively small percentage of human complex I deficiency isassociated with mitochondrial DNA mutations which suggests that most ofthe corresponding mutations affect expression or activity ofnuclear-encoded subunits of complex I.

Scolakin, the protein of the invention, belongs to the category of thenuclear-encoded subunits of complex I. This subunit is part of theiron-sulphur protein fraction (IP) during complex I purification and itis believed to participate to the redox reactions catalysed by theiron-sulphur centers of this complex. NDUFS5 contains four cysteineresidues that form one of the iron-sulphur centers that function inelectron transport. Interestingly, Scolakin contains a fifth cysteinedue to a glycine to cysteine substitution at position 23 on SEQ IDNO:22. Scolakin is widely expressed with a relatively higher expressionin heart, brain, skeletal muscle, liver, kidney and fetal heart andbrain, all tissues with high metabolic activities, which are oftenclinically affected in complex I-deficient patients. In addition,Scolakin expression is high in cancerous tissues and immortalized cellslines, which also have high metabolic requirements. Scolakin plays arole in myopathies, neurodegenerative diseases and cancer.

An embodiment of the invention is directed to a composition comprising aScolakin polypeptide sequence of SEQ ID NO:22.

A further embodiment of the invention is directed to a compositioncomprising a Scolakin polypeptide fragment having biological activity.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:21 encoding a Scolakinpolypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a Scolakin polypeptidefragment having biological activity.

A further embodiment of the invention is directed to a method ofscreening test substances for modulators of Scolakin expressioncomprising the steps of: i) contacting a cell with a substance to betested; and ii) comparing Scolakin expression in the cell after exposureto the test substance to that of an untreated control cell.

In one embodiment, a sequence encoding SEQ ID NO:22 bearing G to T and Cto T substitutions at nucleotide positions 152 and 398 of SEQ ID NO:21corresponding to positions 23 and 105, and resulting in the substitutionof a glycine residue by a cysteine at position 23 and an arginineresidue by a tryptophane at position 105, respectively, can be used forDNA genotyping. Genotyping this locus could be of interest, e.g., in DNAfingerprinting for paternity studies or forensic analyses. It could alsobe used for genetic association studies, preferably in pathologiesrelating to mitochondrial disorders, especially those with isolatedcomplex I deficiency.

In another embodiment, the polynucleotide sequence of the invention canbe used in pharmacogenomic applications in order to aid in the choice ofthe ideal drug (e.g. an agonist or an antagonist of Scolakin), or dosageof a drug, for the treatment of a condition or disease in a patient. Forexample, in one embodiment, the invention provides a method ofgenotyping a patient to determine the identity of the nucleotidesencoding the amino acids at positions 23 and 105 of Scolakin, andadministering to the patient a drug or a dosage of the drug that hasbeen established to be preferentially efficacious in those with cysteineand tryptophane residues at positions 23 and 105, respectively (e.g.because of preferential binding of the drug to the isoform of theprotein with cysteine and tryptophane at these positions). In anotherembodiment, the patient is genotyped for the nucleotides encoding aminoacid positions 23 and 105, a drug is not administered, e.g. because sideeffects are known to be associated with the administration of the drugto individuals with cysteine and tryptophane at positions 23 and 105,respectively.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a Scolakin polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing Scolakinexpression. Preferably, the polynucleotides capable of directingScolakin expression are located in the 5′ regulatory region of theScolakin gene. Further preferably, these polynucleotides are locatedwithin 500 base pairs of the Scolakin coding region. Thesepolynucleotides preferably comprise a promoter sequence. Techniquesknown in the art for introducing polynucleotide sequences to endogenoussequences are described in U.S. Pat. No. 5,641,670 and PCT WO9629411,which disclosures are hereby incorporated by reference in theirentireties. Scolakin protein produced by said host cell may be used forin vitro detection and purification methods as well as diagnosis and invivo applications.

Another embodiment relates to methods of producing Scolakinpolypeptides. The protein of the invention can be produced in host cellsthat have been transfected with an appropriate expression vectorcomprising a nucleic acid sequence coding for the protein of theinvention. Introduction into a host cell of such expression vector forScolakin can be performed in a variety of ways, including but notlimited to calcium or lithium chloride treatment, electroporation, orlipofection. Any of a wide variety of expression systems can be used toprovide the recombinant proteins. Suitable expression vehicles include,but are not limited to plasmids, viral particles or baculovirus forinsect cells. The expression vehicle can be integrated into the hostcell genome. Optionally, an inducible expression vector can be used toachieve tight controlled expression of the gene in the host cell. Therecombinant protein can be recovered from the host cell and purified byany technique well known to those skilled in the art. Preferably, anantibody directed against the protein of the invention or part thereofcan be bound to a chromatographic support to form an affinitychromatography column. Alternatively, Scolakin can be chemicallysynthesized using solid-phase techniques.

In a further embodiment, the invention provides compositions and methodsusing Scolakin or fragments thereof to label mitochondria in order tovisualize any change in number, topology or morphology of thisorganelle, for example in association with a mitochondria-related humandisorder, such as mitochondrial encephalomyopathies, Alpers disease,hereditary spastic paraplegia, neuroleptic malignant syndrome (NMS) orthe Rett syndrome. For example, the protein may be rendered easilydetectable by inserting the cDNA encoding the protein of the inventioninto a eukaryotic expression vector in frame with a sequence encoding atag sequence. A preferred embodiment of the invention is a method todetect mitochondria comprising the step of contacting a monoclonal orpolyclonal antibody specific for Scolakin with a cell. Preferably, thecell is fixed or otherwise permeable to antibodies. Preferably, theantibody is primarily or secondarily linked to a fluorescent,radioactive, or otherwise detectable compound.

In another embodiment, the polynucleotide of SEQ ID NO:21 or fragmentsthereof may be used to target recombinant DNA molecules to themitochondria. For instance, a chimeric polynucleotide composed of thepolynucleotide sequence of the invention recombinantly or chemicallyfused to a polynucleotide of therapeutic interest would allow thedelivery of the therapeutic polynucleotide specifically to themitochondria. Such chimeric molecules would be of particular interest ingene therapy to restore or modulate mitochondrial activities for thetreatment and/or the prevention of disorders due to mitochondrialdysfunction, including, but not limited to, those mentioned above.

In another embodiment, the invention provides compositions and methodsfor detecting the level of expression of the mRNA encoding the proteinof the invention in a mammal, preferably a human. Quantification of mRNAlevels of Scolakin may be useful for the diagnosis of diseases orconditions correlated with abnormal expression of the protein, or tomonitor regulation of Scolakin during therapeutic intervention asdescribed herein. Assays for the detection and quantification of mRNAare well known in the art. Preferred method comprises the steps of:isolating RNA from a biological sample of a subject, measuring ScolakinmRNA level by quantitative RT-PCR, and comparing the expression in thesubject sample to that of a control sample. Polynucleotide probes orprimers used for the detection of Scolakin mRNA by hybridization or PCRamplification may be designed from the polynucleotide of SEQ ID NO:21 bymethods well known in the art.

In another embodiment, the invention relates to methods and compositionsfor detecting Scolakin and quantifying its level of expression in abiological sample. These methods may be useful for the diagnosis ofconditions or diseases characterized by altered or abnormal expressionof the protein of the invention, or in assays to monitor subjects beingtreated with Scolakin, agonists or antagonists. A preferred methodcomprises contacting an antibody which specifically binds to Scolakinwith a biological sample from a mammalian subject, preferably human, anddetermining the level of Scolakin in the subject sample compared to acontrol level representative of a healthy subject, wherein an altered orabnormal level of Scolakin in the subject sample relative to the controllevel indicates that the subject has the disease or is at an elevatedrisk of developing the disease. The antibody used can be eithermonoclonal or polyclonal and can be labeled directly or indirectly forquantification of immune complexes by methods well known to thoseskilled in the art, for example by ELISA or radioimmunoassays.Diagnostic assays to detect the protein of the invention may comprise abiopsy, in situ assay of cells from organ or tissue sections, or anaspirate of cells from a tumor or normal tissue. In addition, assays maybe conducted upon cellular extracts from organs, tissues, cells, urine,or serum or blood or any other body fluid or extract.

Another embodiment of the invention is directed to a method to treat orprevent a defect in mitochondrial complex I of a mammal, preferably ahuman, comprising administering to said mammal the protein of theinvention or fragments thereof or polynucleotides encoding the presentprotein.

Expression vectors derived from retroviruses, adenovirus, herpes, orvaccinia viruses may be used for the delivery of the polynucleotidesequences of the invention to the targeted organ, tissue or cellpopulations. Many methods for introducing vectors into cells or tissuesare available and equally suitable for use either in vivo or ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the subject and clonally propagated for autologous transplant backinto the same subject.

Delivery by transfection and liposome injections may be achieved usingmethods which are well known in the art. Pharmaceutical compositionscomprising Scolakin polypeptides can be administered alone or incombination with at least one other agent, such as stabilizing compound,which may be administered in any sterile biocompatible carrier. They maybe administered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventiculary, transdermal, subcutaneous, intranasal,enteral or rectal means. The compositions may be administered to thesubject alone or in combination with other agents, drugs or hormones.Preferably, the diseases associated with defective complex I activity tobe treated or prevented using the present method include, but are notlimited to, fatal neonatal lactic acidosis, myopathy with exerciseintolerance and lactic acidosis, MELAS (mitochondrial myopathy,encephalopathy, lactic acidosis, and stroke-like episodes), MERFF(myoclonus epilepsy, and ragged red fibres), encephalomyopathy ofchildhood and adult life, Leigh syndrome, Alpers and Parkinson'sdiseases.

An embodiment of the invention provides for a method of screening testsubstances for modulators of Scolakin expression. This method comprisesthe steps of: i) contacting a cell with a test substance; and ii)comparing Scolakin expression in the cell after exposure to the testsubstance to that of an unexposed control cell. Scolakin expression isdetermined by methods common to the art or included herein, by detectingScolakin polynucleotides or polypeptides. An example of this methodcomprises the steps of: i) culturing two equivalent cell samples; ii)adding a test substance to one of the cultures and not the other; iii)harvesting both cultures at a specified time; iv) purifying the mRNAfrom each sample of cells; v) comparing the level of Scolakin mRNA ineach sample by Northern blot, RT-PCR, or another method common to theart. The invention provides for design and use of specificpolynucleotide probes and primers, as discussed herein. An additionalexample comprises the steps of: i) having two equivalent cultures ofcells; ii) adding a test substance to one of the cultures and not theother; iii) harvesting both cultures; iv) purifying the protein fromeach sample of cells; v) comparing the level of Scolakin polypeptides ineach sample by enzyme-linked immunoabsorbent assay (ELISA), westernblot, radioimmunoassay (RIA), or another method common to the art. Theinvention provides for design and use of specific antibodies andantibody fragments, as discussed herein. Substances that increaseScolakin expression (agonists or activators) may be used to increasecomplex I activity. Substances that decrease Scolakin expression(antagonists or inhibitors) may be used to treat or prevent cancers.Methods utilizing Scolakin agonists and antagonists are included herein.

A preferred embodiment of the invention provides a method of screeningfor test substances that bind Scolakin polypeptides. This methodcomprises the steps of: i) contacting a test substance with a Scolakinpolypeptide or fragment thereof under conditions that allow binding; andii) detecting the binding of the test substance by methods common to theart (e.g., competitive antibody-based methods such ascoimmunopreciptation and Western blotting). Included in this method aretest substances that are conjugated to an antibody, antibody fragment,cell-type specific ligand or a portion thereof.

A further preferred embodiment of the invention provides a method ofscreening test substances that bind to Scolakin for agonists of Scolakinactivity, comprising: i) contacting a cell with the substance to betested; and ii) comparing Scolakin biological activity after exposure tothe test substance to that of an unexposed control cell. Measure ofScolakin biological activity may be assessed indirectly by measuring theenzymatic activity of complex I as described by Loeffen et al. (1999, J.Inher. Metab. Dis. Vol. 22, pp 19-28 which disclosure is herebyincorporated by reference in its entirety), an increased in complex Iactivity in the test sample compared to the control sample beingindicative of an activating effect of the test substance on Scolakin.

A further preferred embodiment of the invention provides a method ofscreening test substances that bind to Scolakin for antagonists ofScolakin activity. This method comprises the steps of: i) contacting acell with a test substance; and ii) comparing Scolakin biologicalactivity after exposure to the test substance to that of an unexposedcontrol cell. Detection of Scolakin biological activity may be detectedby measuring complex I activity as mentioned above, a decrease incomplex I activity in the test sample compared to the control samplebeing indicative of an inhibitory effect of the test substance onScolakin activity.

It is yet another object of the invention to provide methods of treatingor preventing cancers associated with increased complex I activity. Thiscan be achieved by decreasing Scolakin expression or activity in vivousing antibodies, ribozymes or antisense vectors or oligonucleotides.Alternatively, an antagonist of Scolakin activity or expression isolatedas described above can be used, such method comprising the step ofadministering to a subject an antagonist of Scolakin expression oractivity. Scolakin antagonists in a physiologically acceptable solutionmay be delivered by methods common to the art, such as orally orparenterally. Preferably, the Scolakin antagonist is delivered to aspecific cell type, for example, by conjugating the antagonist to acell-type specific targeting moiety (e.g., a ligand or antibodyfragment). This method is useful for prevention and treatment of cancersincluding, but not limited to, cancers of the heart, ovaries, colon,kidney, bladder, prostate, pancreas, brain, stomach, breast, lung, liverand leukemias.

Protein of SEQ ID NO:24 (Internal Designation Clone 119658105-067-2-0-H4-F 1)

The cDNA of Clone 119658 105-067-2-0-H4-F_(—)1 (SEQ ID NO:23) encodesthe Docking Of Vesicles (DOV) protein of SEQ ID NO:24, comprising theamino acid sequence:MASRSSDKDGDSVHTASEVPLTPRTNSPDGRRSSSDTSKSTYSLTRRISSLESRRPSSPLIDIKPIEFGVLSAKKEPIQPSVLRRTYNPDDYFRKFEPHLYSLDSNSDDVDSLTDEEILSKYQLGMQHFSTQYDLLHNHLTVRVIEARDLPPPISHDGSRQDMAHSNPYVKICLLPDQKNSKQTGVKRKTQKPVFEERYTFEIPFLEAQRRTLLLTVVDFDKFSRHCVIGKVSVPLCEVDLVKGGHWWKALIPSSQNEVELGELLLSLNYLPSAGRLNVDVIRAKQLLQTDVSQGSDPFVKIQLVHGLKLVKTKKTSFLRGTIDPFYNESFSFKVPQEELENASLVFTVFGHNMKSSNDFIGRIVIGQYSSGPSETNHWRRMLNTHRTAVEQWHSLRSRAECDRVSPASLET. Accordingly, it will beappreciated that all characteristics and uses of the polypeptides of SEQID NO:24 described throughout the present application also pertain tothe polypeptides encoded by the human cDNA included in Clone 119658105-067-2-0-H4-F_(—)1. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:23described throughout the present application also pertain to the nucleicacids comprising the human cDNA in Clone 119658 105-067-2-0-H4-F_(—)1. Apreferred embodiment of the invention is directed toward thecompositions of SEQ ID NO:23, SEQ ID NO:24, and Clone 119658105-067-2-0-H4-F_(—)1. Also preferred are polypeptide fragments having abiological activity as described herein and the polynucleotides encodingthe fragments.

The sequence of DOV contains similarities to proteins of theSynaptotagmin family. Like Synaptotagmin, DOV is a vesiculartransmembrane protein that participates in vesicle docking and fusion. Acalcium-dependent conformational change allows the cytoplasmic portionof DOV to dock with specific proteins on the plasma membrane. Vesicledocking results in release of vesicle contents to the extracellularspace. DOV polypeptides are required for neurotransmitter release from apresynaptic neuron in response to an excitatory calcium flux in thatcell.

A preferred embodiment of the invention includes a compositioncomprising a DOV polypeptide sequence of SEQ ID NO:24.

A preferred embodiment of the invention includes a compositioncomprising a DOV polypeptide fragment having biological activity.

A preferred embodiment of the invention includes a compositioncomprising a polynucleotide sequence of SEQ ID NO:23 encoding a DOVpolypeptide.

A preferred embodiment of the invention includes a compositioncomprising a polynucleotide sequence encoding a DOV polypeptide fragmenthaving biological activity.

A preferred embodiment of the invention includes an excitation-secretionuncoupling peptide (ESUP). Preferred DOV ESUPs include the peptidesequences: LLHNHLTVRVIEARDLPPPISHDGSRQDMAHSNPYVKICLLPDQKNSKQTGVKRKTQKPVFEERYTFEIPFLEAQRRTLLLTVVDFDKFSRHCVIGKS (SEQ ID NO:75);GRLNVDVIRAKQLLQTDVSQGSDPFVKIQLVHGLKLVKTKKTSFLRGTIDPFYNESFSFKVPQEELENASLVFTVFGHNMKSSNDFIGRIVIG (SEQ ID NO:76); and effective ESUPfragments of said peptides.

A preferred embodiment of the invention provides a method of usinginhibitors of DOV polypeptides as anticonvulsants and to prevent pain.These methods comprise the step of administering an effective amount ofa DOV inhibitor in a physiologically acceptable composition to anindividual in need of treatment.

A preferred embodiment provides a method to purify vesicles comprisingthe steps of: i) contacting an antibody specific for the cytoplasmicportion of DOV polypeptide with a biological sample under conditionsthat allow antibody binding and ii) removing contaminating materials notbound to the antibody. Preferably, such antibody is primarily orsecondarily attached to an insoluble matrix to enable purification.

A preferred embodiment provides a method to detect vesicles comprisingthe step of contacting an antibody specific for DOV polypeptide with abiological sample under conditions that allow binding and ii) detectingthe antibody. Preferred antibodies are covalently labeled withfluorescent, radioactive, or otherwise detectable compounds.

A preferred embodiment provides a method to detect vesicle extrusioncomprising the step of contacting an antibody specific for the vesicularportion of DOV with a cell. Preferred antibodies are covalently labeledwith fluorescent, radioactive, or otherwise detectable compounds.Preferred cells are neurons.

A preferred embodiment of the invention provide a method to diagnose aneurological disorder comprising the steps of: obtaining a biologicaltest sample from an individual; detecting the level of DOV expression inthe test sample; comparing to the level of DOV expression in the testsample to that of control sample(s). Neurological disorders indicated bythis method include: Paroxysmal Kinesigenic Choreoathetosis (PKC),paralysis (partial and complete), and mood disorders (depression,bipolar disorder, and schizophrenia).

A preferred embodiment is a method to restore vesicular docking andfusion abilities to a cell comprising the step of introducing DOVpolypeptides to a cell. Preferably, this method is applied to treatmentand prevention of PKC, partial and complete paralysis, and mooddisorders such as depression, bipolar disorder, and schizophrenia.Preferably, DOV polypeptides are delivered by introducing apolynucleotide construct comprising an expression control elementoperably linked to polynucleotide encoding a DOV polypeptide to a cell.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a DOV polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing DOV expression.Preferably, the polynucleotides capable of directing DOV expression arelocated in the 5′ regulatory region of the DOV gene. Further preferably,these polynucleotides are located within 500 base pairs of the DOVcoding region. These polynucleotides preferably comprise a promotersequence. Techniques known in the art for introducing polynucleotidesequences to endogenous sequences are described in U.S. Pat. No.5,641,670 and PCT WO9629411, which disclosures are hereby incorporatedby reference in their entireties. DOV protein produced by said host cellmay be used for in vitro detection and purification methods as well asdiagnosis and in vivo applications.

In one aspect, the invention includes a DOV polypeptide composition foruse in delivering a second composition, preferably nucleic acids,polypeptides, or small molecules, as therapeutic drugs, to targetbiological cells either in vitro or in vivo. The second composition may,if desirable, be covalently or non-covalently attached or fused to theDOV polypeptide. The DOV polypeptide composition may further compriseartificial lipids or viral components to facilitate delivery of thesecond molecule by liposomes or lipid vesicles. Methods for using DOVpolypeptides in these methods are known in the art and include U.S. Pat.Nos. 6,074,844 and 6,099,857, which disclosures are hereby incorporatedby reference in their entireties. In a preferred embodiment, DOVpolypeptides are used to faciliate delivery of a second composition,e.g., liposome-mediated DNA transfection, to cells in culture,preferably neuronal cells.

DOV polypeptides are also useful in methods of inhibiting the release ofneurotransmitters by preventing the docking and/or fusing of apresynaptic vesicle to the presynaptic membrane. These polypeptides maybe referred to as excitation-secretion uncoupling peptides (ESUPs).Fragments of DOV having this blocking activity can be identified usingmethods known in the art (See e.g., U.S. Pat. Nos. 6,090,631 and6,169,074 incorporated by reference in their entireties). ESUPs of thepresent invention comprise synthetic and purified DOV peptide fragmentswhich correspond in primary structure to peptides which serve as bindingdomains for the assembly of a ternary protein complex (“dockingcomplex”) which is critical to neuronal vesicle docking with thecellular plasma membrane prior to neurotransmitter secretion. Foroptimal activity, ESUPs of the invention have a minimum length of about20 amino acids and a maximal length of about 100 amino acids, althoughthey may be larger or smaller. Preferred DOV ESUPs for use in inhibitingthe release of neurotransmitters include those comprising the sequence:LLHNHLTVRVIEARDLPPPISHDGSRQDMAHSNPYVKICLLPDQKNSKQTGVKRKTQKPVFEERYTFEIPFLEAQRRTLLLTVVDFDKFSRHCVIGKS (SEQ ID NO:75);GRLNVDVIRAKQLLQTDVSQGSDPFVKIQLVHGLKLVKTKKTSFLRGTIDPFYNESFSFKVPQEELENASLVFTVFGHNMKSSNDFIGRIVIG (SEQ ID NO:76); and effective ESUPfragments of said peptides. DOV ESUPs are preferably covalently linkedto a targeting moiety. Preferred targeting moieties comprise a ligand toa cell-surface binding site present on a specific cell type (e.g.,neuron) that is capable of functionally interacting with the bindingsite. Preferred targeting moieties are nerve growth factor andfunctional derivatives thereof. Alternatively, DOV ESUPs may beexpressed in a cell by introducing polynucleotides encoding DOV ESUPs tothe cell.

Methods of introducing polynucleotides to a cell are known to thoseskilled in the art, as discussed herein and in U.S. Pat. Nos. 6,117,454and 6,180,602, which disclosures are hereby incorporated by reference intheir entireties.

Another embodiment of the invention provides a method of screening forantagonists of DOV polypeptides. This method comprises the steps of:contacting a test substance with a cell and detecting DOV biologicalactivity in the exposed cell to that in an unexposed control cell.Preferred cells are neurons. DOV polypeptides are required for efficientneurotransmitter release. Thus, DOV activity can be determined bydetecting the level of neurotransmitter released from exposed cellscompared to that from unexposed cells. Such assays are common to the artand include antibody-based assays (e.g., ELISA), binding competitionassays (e.g., to a specific neurotransmitter receptor), and colormetricassays. Preferred antagonists include DOV-specific antibodies andfragments thereof, ESUPs, and small molecules.

DOV antagonists may be used to inhibit or treat pain. Peptideantagonists in particular may be used according to U.S. Pat. No.5,989,545 (disclosure of which is hereby incorporated by reference inits entirety) by substituting the polypeptides of the present inventionfor neurotoxin. In addition, DOV antagonists of the invention may beused as anticonvulsants. Anticonvulsants effectively reduce infarct sizein cases of stroke and are effective therapy for seizures, such as thoseresulting from epilepsy or exposure to neurotoxins. These methodscomprise the step of administering an effective amount of DOV antagonistin a physiologically acceptable composition to an individual in need oftreatment.

DOV is a component of vesicles, thus, antibodies to DOV are useful inthe detection of vesicles, preferably neuronal vesicles transportingneurotransmitters. DOV-specific antibodies can be used duringpurification of vesicles. This method comprises the steps of: i)contacting an antibody specific for the cytoplasmic portion of DOVpolypeptide with a biological sample under conditions that allowantibody binding and ii) removing contaminating materials not bound tothe antibody. Preferably, such antibody is primarily or secondarilyattached to an insoluble matrix to enable purification. DOV-specificantibodies may also be used as a marker for vesicles. For example,vesicles can be detected in assays such as immunohistochemistry. Thevesicular portion of DOV appears on the surface of the cell followingextrusion of vesicle contents at the plasma membrane. Thus, antibodiesspecific for the vesicular portion of DOV are useful for the detectionand monitoring of vesicle extrusion. This method comprises the step ofcontacting an antibody specific for the vesicular portion of DOV with acell. Preferred antibodies are detectably labeled, for example, with afluorescent, luminescent, or radiolabeled compound.

Detection of DOV expression (mRNA or protein) levels or mutated forms ofDOV is further useful in the determination or diagnosing of whethersomeone is at risk of developing or has a neurological disorder, such asParoxysmal Kinesigenic Choreoathetosis (PKC), paralysis (partial orcomplete), or mood disorders (e.g., depression, bipolar disorder,schizophrenia). This method comprises the steps of: obtaining abiological test sample from an individual; detecting the level of DOVexpression in the test sample; comparing to the level of DOV expressionin the test sample to that of control sample(s). A decreased level inexpression of DOV, mRNA or protein, as compared to an individual withouta neurological disorder indicates the individual has or is as risk ofdeveloping the disorder.

DOV polypeptides may further be introduced to a cell that is defectivein vesicle docking and fusion. This method comprises the step ofintroducing DOV polypeptides to a cell such that vesicle docking andfusion are allowed. DOV polypeptides may be introduced to a cell byintroducing a polynucleotide construct comprising an expression controlelement operably linked to the coding sequence of DOV to a cell bymethods discussed herein. Preferred cells are neurons. DOV polypeptidesare preferably applied to treatment of PKC, paralysis (partial orcomplete), or mood disorders (e.g., depression, bipolar disorder,schizophrenia).

Protein of SEQ ID NO:26 (Internal Designation Clone500706437_(—)204-5-2-0-C9-F)

The cDNA of Clone 500706437_(—)204-5-2-0-C9-F (SEQ ID NO:25) encodesplacentalin of SEQ ID NO:26, comprising the amino acid sequence:MDPARPLGLSILLLFLTEAALGDAAQEPTGNNAEICLLPLDYGPCRALLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDLAGG. Accordingly, it will be appreciated that allcharacteristics and uses of the polypeptides of SEQ ID NO:26 describedthroughout the present application also pertain to the polypeptidesencoded by the human cDNA included in Clone 500706437_(—)204-5-2-0-C9-F.In addition, it will be appreciated that all characteristics and uses ofthe polynucleotides of SEQ ID NO:25 described throughout the presentapplication also pertain to the nucleic acids comprising the human cDNAin Clone 500706437_(—)204-5-2-0-C9-F. A preferred embodiment of theinvention is directed toward the compositions of SEQ ID NO:26, SEQ IDNO:25, and Clone 500706437_(—)204-5-2-0-C9-F. Preferred placentalinpolypeptides for uses in the methods described below include thepolypeptides comprising the amino sequence of:AQEPTGNNAEICLLPLDYGPCRALLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDD LAGG (SEQID NO:77). Also preferred are polypeptide fragments having a biologicalactivity as described herein and the polynucleotides encoding thefragments.

The protein of the present invention, placentalin, is a variant of theTissue Factor Pathway Inhibitor 2 (TFPI-2, GenBank accession numberP48307). The 84 amino-terminal amino-acids are identical between TFPI-2and placentalin, and the 4 carboxyl-terminal amino-acids are unique toplacentalin. Placentalin displays one signal peptide(MDPARPLGLSILLLFLTEAALGDA) (SEQ ID NO:78) and one Kunitz inhibitordomain (CLLPLDYGPCRALLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDLA) (SEQ IDNO:79). Placentalin lacks the main heparin-binding site, located at thecarboxyl-terminal extremity of TFPI-2. A wide variety of cell types,including endothelial cells, keratinocytes and fibroblasts, synthesizeand secrete placentalin under normal conditions. Moreover, placentalinis highly expressed in umbilical vein endothelial cells and itsexpression level negatively correlates with malignancy-grade of tumors.

Placentalin is a broad-spectrum serine protease inhibitor that inhibitsplasmin, trypsin, plasma kallikrein, factor XIa, tissue factor-factorVIIa complex, chymotrypsin, factor IXa-polylysine and cathepsin G.Placentalin plays a major role both in regulation of extracellularmatrix (ECM) degradation and in regulation of the blood coagulationpathway. In peculiar, placentalin strongly inhibits the tissuefactor-factor VIIa complex, the cellular initiator of the extrinsiccoagulation pathway, and plasmin, thereby inhibiting processing ofpro-MMPs (matrix metalloproteases) responsible of ECM degradation.Unlike TFPI-2, placentalin is a circulating variant that is not bound tothe heparin molecules of the ECM.

An embodiment of the invention is directed to a composition comprising aplacentalin polypeptide sequence of SEQ ID NO:26.

A further embodiment of the invention is directed to a compositioncomprising a placentalin polypeptide fragment having biological activityof binding or inhibiting a serine protease.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:25 encoding aplacentalin polypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a placentalin polypeptidefragment having biological activity.

A further embodiment of the invention is directed to a compositioncomprising an antibody recognizing a placentalin polypeptide sequence ofSEQ ID NO:26 or a placentalin polypeptide fragment having biologicalactivity. Preferably, the antibody recognizes an epitope comprising oneor more of the 4 C-terminal amino-acids of placentalin, and binds toplacentalin but not to TFPI-2.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a placentalin polypeptide or a biologicallyactive fragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing placentalinexpression. Preferably, the polynucleotides capable of directingplacentalin expression are located in the 5′ regulatory region of theplacentalin gene. Further preferably, these polynucleotides are locatedwithin 500 base pairs of the placentalin coding region. Thesepolynucleotides preferably comprise a promoter sequence. Techniquesknown in the art for introducing polynucleotide sequences to endogenoussequences are described in U.S. Pat. No. 5,641,670 and PCT WO9629411,which disclosures are hereby incorporated by reference in theirentireties. Placentalin protein produced by said host cell may be usedfor in vitro detection and purification methods as well as diagnosis andin vivo applications.

Another embodiment relates to a method of producing placentalinpolypeptides comprising the steps of: i) transfecting a mammalian hostcell with a recombinant expression vector comprising a polynucleotide ofthe present invention, and ii) purifying the produced protein. Thepurification of the protein can be done following any techniquewell-known to those skilled in the art. Preferably, an antibody directedagainst placentalin or part thereof may be bound to a chromatographicsupport to form an affinity chromatography column. Even more preferably,the antibody binds to placentalin but not to TFPI-2.

A preferred embodiment of the invention is a method of using placentalinto bind serine proteases. This method comprises the step of contacting aplacentalin polypeptide or active fragment thereof with a serineprotease under conditions that allow placentalin binding, wherebybinding inhibits the activity of said serine protease. Preferred serineproteases include plasmin, trypsin, plasma kallikrein, factor XIa,tissue factor-factor VIIa complex, chymotrypsin, factor IXa-polylysineor cathepsin G. This method may be applied to in vitro uses (e.g.,protease purification and detection and prevention of proteindegradation) as well as in vivo methods (e.g., inhibition of targetprotease-dependent pathologies such as blood clots and metastasis).

A preferred embodiment of the invention is a method of purifying serineproteases. This method comprises the steps of: contacting a placentalinpolypeptide or protease-binding fragment thereof with a serine proteaseunder conditions that allow binding; removing contaminants; and elutingthe serine protease with more stringent conditions. Preferably, theplacentalin peptide is immobilized on a solid or semi-solid matrix tofacilitate washing of sample to remove contaminants. Preferred serineproteases include plasmin, trypsin, plasma kallikrein, factor XIa,tissue factor-factor VIIa complex, chymotrypsin, factor IXa-polylysineor cathepsin G. These may be purified from common biological fluids suchas cell culture media and body fluids (e.g., serum, blood, lymph, orcerebrospinal fluid). Purified proteases are useful in biological assaysand techniques such as removal of adherent cells from a culture dish andsite-directed peptide design.

A further embodiment of the present invention relates to a method ofusing placentalin or a protease-binding fragment thereof to removecontaminating proteases from a sample comprising the steps of: i)binding placentalin or part thereof to a chromatographic support, eitheralone or in combination with other protease inhibitors, and ii) runningthrough the column the sample containing the undesirable protease. Thesample may be, e.g., a protein preparation or a sample prepared frombodily fluid. Preferably, the sample is prepared from blood.

An embodiment of the present invention relates to a method of usingplacentalin or part thereof to inhibit contaminating proteases in asample. Such method comprises the step of adding a protease-inhibitingamount of placentalin polypeptide to a solution under conditions thatallow placentalin activity. Preferably, the placentalin polypeptide iscomprised in a “cocktail” of protease inhibitors that is able to inhibita wide range of proteases without knowing the specificity of any of theproteases. Such protease inhibitor cocktails are widely used inlaboratory assays to prevent degradation of any protein sample bycontaminating proteases.

A preferred embodiment of the inventions is a method of detecting serineproteases. This method comprises the step of contacting a placentalinpolypeptide or active fragment thereof with a serine protease anddetecting the presence of said serine protease by detecting placentalin.Preferably, the placentalin polypeptide is detectably labeled with, forexample, a fluorescent, luminescent, or radioactive compound. Preferredserine proteases to be detected according to this method includeplasmin, trypsin, plasma kallikrein, factor XIa, tissue factor-factorVIIa complex, chymotrypsin, factor IXa-polylysine or cathepsin G.Preferably, serine proteases are detected in common biological fluidssuch as cell culture media and body fluids. This method may be appliedto quantifying the level of serine protease expression in a cell sampleor individual. This information may be useful in determination of tumormalignancy or diagnosis of blood coagulation disorders.

An additional preferred embodiment of the invention is a method ofbinding placentalin polypeptides with a placentalin-specific antibody orplacentalin-binding fragment thereof. This method comprises the step ofcontacting a placentalin polypeptide with a placentalin-binding antibodyor placentalin-binding fragment thereof under conditions that allowbinding. This method may be applied to detection and purification ofplacentalin, as well as modifying placentalin function. These aspectsare discussed in detail herein.

Another embodiment of the present invention relates to a method ofdiagnosing the malignancy of a tumor comprising the steps of: i)contacting a biological sample with a compound that specifically bindsto placentalin polypeptides or nucleic acids, ii) detecting the level,or any other detectable property, of placentalin in the sample, and iii)comparing the resulting signal with those obtained from control samplesindicative of a benign, a low-grade, an intermediate-grade and ahigh-grade malignant tumor. The level of the signal negativelycorrelates with the malignancy of the tumor. Preferably, this method isdirected to diagnose the malignancy of tumors such as, e.g., gliomas,amelanotic melanomas, prostate tumors and lung tumors.

Still another embodiment of the present invention relates to a method ofdiagnosing the risk for thrombotic deposition comprising the steps of:i) contacting a biological sample with a compound that specificallybinds to placentalin polypeptides or nucleic acids, ii) detecting thelevel, or any other detectable property, of placentalin in the sample,and iii) comparing the resulting signal with those obtained from controlsamples indicative of a patient presenting no risk for thromboticdeposition. Preferably, such methods are directed to diagnosehypercoagulable syndromes, e.g., artherosclerosis, ischemia, stroke,myocardial infarction, coronary artery disease.

Preferably, the biological sample is either a body fluid or tissuesample that has been purified or partially purified to ease detection.Particularly preferred compounds that specifically bind to placentalinpolypeptides are the antibodies described above. Preferably, theantibodies are labeled and detection of the signal may be carried outusing immunohistological and immunofluorescing processes that arewell-known to those skilled in the art. Alternatively, the antibodiesthat specifically bind to placentalin polypeptides are not labeled anddetection of the signal occurs indirectly by using labeled secondaryantibodies. Particularly preferred compounds that specifically bind toplacentalin polynucleotides are polynucleotide sequences that arecomplementary to SEQ ID NO:25 or part thereof. Detection of the signalmay be carried out using PCR, RT-PCR techniques, or quantitative PCRtechniques.

Still another embodiment relates to a kit for determining the malignancyof a tumor or the risk for thrombotic deposition. Such kits comprise atleast one binding partner specific for placentalin polypeptides ornucleic acids coupled to a detectable marker, control samples, andreagents for performing the reaction for detecting the marker.Preferably, the kit is directed to diagnosing the malignancy of a giventumor, e.g., gliomas, amelanotic melanomas, prostate tumors and lungtumors. Alternatively, the kit is directed to monitor the progress of atherapy administered to treat malignant tumors.

Another preferred embodiment relates to substances that increaseplacentalin expression and to a method of screening for substances thatincrease placentalin expression comprising the steps of:

i) contacting a cell with a test substance; and ii) comparingplacentalin expression in the cell after exposure to the test substanceto that of an unexposed control cell. Preferably, the test substancemodifies the expression of placentalin in a specific cell type while notin others. Most preferably, the test substance modifies placentalinexpression specifically in endothelial cells or in neoplastic cells.

A further embodiment of the present invention is directed to substancesthat increase placentalin activity and to a method of screening for suchsubstances. This method comprises the steps of: i) contacting aplacentalin polypeptide with a test substance, ii) determiningplacentalin activity, and iii) comparing placentalin activity afterexposure to that of an unexposed control sample. Preferably, placentalinactivity is studied in an endothelial cell or in a neoplastic cell.Placentalin activity can be monitored by studying plasmin inhibitionand/or radiolabeled matrix degradation as described in Rao et al.(Biochem Biophys Res Commun. 276:1286-94 (2000)), which disclosure isincorporated herein in its entirety. Substances that increaseplacentalin expression or activity are defined as placentalin agonists.

An embodiment of the present invention relates to methods of using thepolypeptides and the polynucleotides of the present invention to reduceblood coagulation in a mammal. Any compositions and methods containing aplacentalin polypeptide or a placentalin agonist can be used.Preferably, such methods for reducing blood coagulation are directed totreat a patient presenting a risk for thrombotic deposition. Mostpreferably, such methods are directed to treat a patient suffering fromthrombotic diseases and/or hypercoagulable syndromes, e.g.,artherosclerosis, acute ischemia, stroke, myocardial infarction,coronary artery disease, and thrombotic complications associated withtransplantation. A preferred embodiment is a method to reduce bloodcoagulation comprising the step of introducing a placentalin polypeptideor a protease-inhibiting fragment thereof in a physiologicallyacceptable composition to the bloodstream of an individual. Delivery tothe bloodstream can be direct (e.g., injection to a vein or artery) orindirect (e.g., inhalant or oral delivery), as discussed in detailherein.

In a further embodiment, the present invention provides a method ofpromoting wound healing by preventing tissue buildup using compositionscomprising a placentalin polypeptide or a placentalin agonist. Such amethod comprises the step of: contacting said wound with an effectiveamount of the composition comprising a placentalin polypeptide or aplacentalin agonist, wherein the wound includes, but is not limited to,myocardial infarction, skin wound, arterial wall injury, stents andsutures.

Another embodiment of the present invention relates to methods of usingthe polypeptides and the agonists of the present invention to prevent orreduce ECM degradation in a mammal.

Preferably, such methods for preventing or reducing ECM degradation aredirected to protect the ECM from degradation by malignant cells, thusblocking the invasion and spread of malignant tumors. Most preferably, acomposition comprising a placentalin polypeptide is administered to anindividual suffering from abnormal or undesirable cell proliferation,such as, e.g., tumor growth, endothelial cell proliferation, andangiogenesis related to tumor growth. Tumors that can be treated withthe compositions of the present invention include, but are not limitedto, gliomas, amelanotic melanomas, prostate tumors and lung tumors.

Still another embodiment of the invention relates to the inhibitionand/or attenuation of proteases produced by pathogenic microorganisms.This embodiment relates to a method comprising the step of administeringa composition comprising placentalin or a biologically active fragmentthereof, or a compound that increases the expression or activity ofplacentalin, to an individual for preventing and/or treating parasiticinfections. Placentalin polypeptides or compounds that increase theexpression or activity of placentalin may be administered alone or incombination with other agents. Alternatively, this method may bedirected to prevent parasitic infections of mammalian cell cultures. Ithas been shown that protease inhibitors can prevent dissemination ofvirus, protozoa, bacteria or fungi in the host organism. Accordingly,the polypeptides of the present invention may be used to prevent or totreat, e.g., coccidiosis, staphylococcal infection, infection by theinfluenza virus, P. gingivalis or T. denticola, and invasive pulmonaryaspergillosis.

In one embodiment, the pharmaceutical compositions for use in preventionof ECM degradation or inhibition of blood coagulation comprise aneffective amount of a placentalin polypeptide, placentalinpolynucleotide, or substance that increases placentalin expression oractivity in mixture with a pharmaceutically acceptable diluent orcarrier. An effective amount of the compositions of the presentinvention may vary according to factors such as the disease, the diseasestate, age, sex, and weight of the mammal. Dose regimen may be adjustedto provide the optimum therapeutic response. In a preferred embodiment,a composition comprising a substance that increases placentalinexpression or activity and a physiologically acceptable carrier isdirectly introduced to the bloodstream of an individual by injection.

In a preferred embodiment, a placentalin polypeptide is fused to atargeting molecule specific for the tissue of interest. Preferably, thetargeted placentalin polypeptide is comprised in a pharmaceuticalcomposition for use in prevention of ECM degradation associated withtumor growth. Most preferably, the placentalin polypeptide is linked toa ligand or an antibody that recognizes a receptor or an antigenspecifically expressed on tumor cells by any technique well-known tothose skilled in the art. A large number of tumor-associated antigenshave been described in the scientific literature. For example, antigensand techniques described by Wikstrand et al (Cancer Metastasis Rev18:451-64 (1999)), which disclosure is hereby incorporated by referencein its entirety, may be used. Alternatively, the placentalin polypeptideis linked to a ligand that recognizes the vasculature of a tumor. Apreferred method for targeting the placentalin polypeptide to thevasulature of solid tumors is described in U.S. Pat. No. 6,004,554,which disclosure is incorporated by reference in its entirety.

Another embodiment is directed to a method of using placentalinpolynucleotides for reducing blood coagulation comprising the steps of:i) constructing a recombinant molecule comprising a nucleic acidsequence encoding a placentalin polypeptide that allows expression ofplacentalin or part thereof under given physiological conditions, andii) introducing this recombinant molecules into a cell, a mammal or ahuman. Recombinant molecules comprising a nucleic acid sequence encodinga placentalin polypeptide may be directly introduced into cells ortissues in vivo using delivery vehicles such as retroviral vectors,adenoviral vectors and DNA virus vectors. They may also be introducedinto cells in vivo using physiological techniques such as microinjectionand electroporation or chemical methods such as coprecipitation andincorporation of DNA into liposomes. Recombinant molecules may also bedelivered in the form of an aerosol or by lavage. The placentalinpolynucleotides may also be applied extracellularly such as by directinjection into cells. Preferably, placentalin polynucleotides areintroduced into endothelial cells or in blood vessel cells. Mostpreferably, when placentalin polynucleotides are directed to treating apatient presenting a risk for thrombotic deposition, the methoddescribed in PCT application WO 99/02171, which disclosure isincorporated by reference in its entirety, can be used.

Protein of SEQ ID NO:28 (Internal Designation Clone 176355117-005-2-0-H11-F)

The cDNA of Clone 176355_(—)117-005-2-0-H11-F (SEQ ID NO:27) encodesNAAR protein of SEQ ID NO:28, comprising the amino acid sequence:MASMAAVLTWALALLSAFSATQARKGFWDYFSQTSGDKGRVEQIHQQKMAREPATLKDSLEQDLNNMNKFLEKLRPLSGSEAPRLPQDPVGMRRQLQEELEEVKARLQPYMAEAHELVGWNLEGLRQQLKPYTMDLMEQVALRVQELQEQLRVVGEDTKAQLLGGVDEAWALLQGLQSRVVHHTGRFKELFHPYAESLVSGIGRHVQELHRSVAPHAPASPARLSRCVQVLSRKLTLKAKALHARIQQNLDQLREELSRAFAGTGTEEGAGPDPQMLSEEVRQRLQAFRQDTYLQIAAFTRAIDQETEEVQQQLAPPPPGHSAFAPEFQQTDSGKVLSKLQARLDDLWEDITHSLHDQGH SHLGDP.Accordingly, it will be appreciated that all characteristics and uses ofthe polypeptides of SEQ ID NO:28 described throughout the presentapplication also pertain to the polypeptides encoded by the human cDNAincluded in Clone 176355_(—)117-005-2-0-H11-F. In addition, it will beappreciated that all characteristics and uses of the polynucleotides ofSEQ ID NO:27 described throughout the present application also pertainto the nucleic acids comprising the human cDNA in Clone176355_(—)117-005-2-0-H11-F. A preferred embodiment of the invention isdirected toward the compositions of SEQ ID NO:27, SEQ ID NO:28 and Clone176355_(—)117-005-2-0-H11-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

The protein of the invention displays an Apolipoprotein motif:

MAAVLTWALALLSAFSATQARKGFWDYFSQTSGDKGRVEQIHQQKMAREPATLKDSLEQDLNNMNKFLEKLRPLSGSEAPRLPQDPVGMRRQLQEELEEVKARLQPYMAEAHELVGWNLEGLRQQLKPYTMDLMEQVALRVQELQEQLRVVGEDTKAQLLGGVDEAWALLQGLQSRVVHHTGRFKELFHPYAESLVSGIGRHVQELHRSVAPHAPASPARLSRCVQVLSRKLTLKAKALHARIQQNLD (SEQ ID NO:80). Accordingly, an embodiment of thepresent invention comprises the amino acids of the apolipoprotein motifand the polynucleotides encoding the same.

Lipoprotein particles such as HDL and LDL contain characteristicapolipoproteins that are responsible for targeting them to certaintissues and for activating enzymes required for the trafficking of thelipid fraction of the lipoprotein, including cholesterol. NAAR is amember of the apolipoprotein family.

Apolipoproteins are the protein component of lipoprotein particles andare responsible for binding to receptors on cell membranes and directingthe lipoprotein particles to their intended site of metabolism. Thesehigh molecular weight particles are primarily responsible for lipidtransport (triglycerides and cholesterol in the form of cholesterylesters) through the plasma. Lipoprotein particles include chylomicronsand chylomicrons remnant particles, very low density lipoprotein (VLDL),intermediate density lipoprotein (IDL), low density lipoprotien (LDL),and high density lipoprotein (HDL). Elevated levels of lipoproteinparticles have been positively correlated with artherosclerosis and areduction in plasma lipoprotein concentration is correlated with areduced risk of artherosclerosis. In addition, individualapolipoproteins have unique functions such as the formation of specificassociations with lipoprotein particles of distinct classes. Someapolipoproteins act as ligands controlling the interaction oflipoproteins with cell surface receptors, while others functions ascofactors for essential enzymes in lipid metabolism.

NAAR is associated with the chylomicron and HDL fraction of blood. NAARsynthesis by the small intestine increases markedly after the ingestionof lipid with the result being a marked increase in NAAR output inmesenteric lymph. During secretion from small intestine epithelialcells, the pre-NAAR signal peptide is cleaved. The mature NAARpolypeptide is secreted into the lymph as a major constituent of newlysynthesized triglyceride-rich lipoprotein as well as the HDL fraction ofblood. NAAR is involved in the biogenesis and/or metabolism ofintestinal triglyceride-rich lipoproteins. This increase in biosynthesisand secretion of NAAR by the small intestine is triggered by theformation and secretion of intestinal chylomicrons. NAAR levels areregulated by diet and are up-regulated by high fat meals anddown-regulated by leptins. NAAR in mesenteric lymph after a lipid mealsuppresses food intake, suggesting that NAAR also acts as a satietyfactor that circulates in the blood after fat feeding.

NAAR plays a role in triglyceride-rich lipoprotein metabolism, inreverse cholesterol transport, and in facilitating CETP (CholesterolEster Transfer Protein) activity. As a result, NAAR is responsible forpart of the inter-individual variability in blood cholesterol responseto changes in dietary fat/cholesterol intake. Moreover, NAAR may beeffectively used in place of HDL particles to prevent the development ofatherosclerosis. Also, NAAR plays a significant role in thepathophysiology of diabetes. Levels of NAAR are correlated with glycemiccontrol in young type I diabetes (IDDM) patients andnon-insulin-dependent diabetes melitus (NIDDM) patients.

NAAR plays a major role in lipid metabolism and its related disorders.Diseases involving lipid metabolism include, but are not limited to,obesity, obesity-related disorders such as artherosclerosis,cardiovascular disorders such as coronary heart disease,neurodenegenrative disorders such as Alzheimer's disease or dementia,coronary artery disease, mitochondriocytopathies, hyperlipidemia,familial combined hyperlipidemia (FCHL), hypercholesterolemia,obesity-related artherosclerosis, obesity-related insulin resistance,obesity related hypertension, microangiopathic lesions resulting fromobesity-related Type II diabetes, ocular lesions caused bymicroangiopathy in obese individuals with type II diabetes, and renallesions caused by microangiopathy in obese individuals with type IIdiabetes.

An embodiment of the invention is directed to a composition comprising aNAAR polypeptide sequence of SEQ ID NO:28.

A further embodiment of the invention is directed to a compositioncomprising a NAAR polypeptide fragment having biological activity.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:27 encoding a NAARpolypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a NAAR polypeptidefragment having biological activity NAAR polypeptides bind free fattyacids (FFAs), triglycerides, and cholesterol, which are associated withatherosclerosis and lipid metabolism-related diseases. A preferredembodiment of the invention is a method comprising the step of: bindinga NAAR polypeptide or fragment thereof with an FFA, triglyceride, orcholesterol molecule under conditions that allow binding of NAARpolypeptide to said molecule. In a preferred embodiment, NAARpolypeptide is used to purify FFAs, triglycerides, and cholesterol. Insuch method, NAAR polypeptide is preferably covalently or non-covalentlyattached to a solid matrix and allowed to bind FFAs, triglycerides, andcholesterol using techniques well known in the art. This methodcomprises the steps of: i) washing the solid matrix to get rid ofcontaminants, ii) eluting the particle of interest using more stringentconditions.

Additional aspects of this embodiment include methods of using NAARpolypeptide to detect and quantify FFAs, triglycerides, and cholesterolusing techniques common in the art. This method comprises the steps of:i) obtaining a biological sample suspected of containing FFAs,triglycerides, or cholesterol; ii) contacting said sample with a NAARpolypeptide or fragment thereof under conditions suitable for binding ofNAAR; and detecting the presence or absence of FFAs, triglycerides, andcholesterol by detecting NAAR. Preferably, NAAR polypeptide or fragmentthereof is covalently attached to a detectable compound. Alternatively,a detectable NAAR-specific antibody or fragment thereof may be used todetect NAAR. This embodiment is useful, for example, as a diagnostictool for detecting plasma levels of FFAs, triglycerides, andcholesterol.

In another embodiment, the invention is directed to a method ofdetecting NAAR polypeptide in a biological sample, said methodcomprising the steps of: i) contacting a biological sample with anantibody or antibody fragment that specifically binds NAAR polypeptide;and ii) detecting the antigen-antibody complex formed. The antibody orantibody fragment may be monoclonal or polyclonal. In addition, theantibody or antibody fragment may be primarily or secondarily labeled bya detectable compound (e.g., radioactive, fluorescent, luminescent, orenzymatic) common in the art.

In some embodiments, the invention also concerns a diagnostic kit fordetecting in vitro the presence of NAAR polypeptide. This kit comprises:a polyclonal or monoclonal antibody or fragment thereof thatspecifically binds a NAAR polypeptide; and optionally, ii) a reagentallowing the detection of the antigen-antibody complexes formed.Preferably, the antibody or antibody fragment is detectably labeled.Such labels include fluorescent, luminescent, and radioactive compounds,as well as enzymatic substrates. The optional reagent may provide adetectable signal and either bind to the antibody or react with thelabel on such antibody. NAAR antibodies may be used to diagnoseliver-related disorders (e.g., hepatitis, cirrhosis, hepatoma, and FHP),lipid metabolism related disorders such as diabetes, andatherosclerosis. To detect such disorders, an appropriate biologicalsample (serum, for example) can be tested to determine the level of NAARbeing produced (U.S. Pat. No. 6,027,935, which disclosure isincorporated by reference in its entirety). (U.S. Pat. No. 6,027,935,which disclosure is incorporated by reference in its entirety).

An embodiment of the invention provides for a method of screening testsubstances for modulators of NAAR expression. This method comprises thesteps of: i) contacting a cell with a test substance; and ii) comparingNAAR expression in the cell after exposure to the test substance to thatof an unexposed control cell. NAAR expression is determined by methodscommon to the art or included herein, by detecting NAAR polynucleotidesor polypeptides. An example of this method comprises the steps of: i)culturing two equivalent cell samples; ii) adding a test substance toone of the cultures and not the other; iii) harvesting both cultures ata specified time; iv) purifying the mRNA from each sample of cells; v)comparing the level of NAAR mRNA in each sample by Northern blot, RTPCR,or another method common to the art. The invention provides for designand use of specific polynucleotide probes and primers, as discussedherein. An additional example comprises the steps of: i) having twoequivalent cultures of cells; ii) adding a test substance to one of thecultures and not the other; iii) harvesting both cultures; iv) purifyingthe protein from each sample of cells; v) comparing the level of NAARpolypeptides in each sample by Western blot, immunohistochemistry, oranother method common to the art. The invention provides for design anduse of specific antibodies and antibody fragments, as discussed herein.

In another embodiment, a NAAR polypeptide or a fragment thereof, may beused to screen for compounds that activate or inhibit NAAR activity.This method comprises the steps of: i) contacting a NAAR polypeptide orfragment thereof with a test substance and ii) monitoring NAAR activity.NAAR binds to bind FFAs, triglycerides, and cholesterol. Thus, NAARactivity may be monitored upon addition of the test substance bycompetitive binding assays with FFAs, triglycerides, or cholesterol. Inthis aspect of the invention, a NAAR polypeptide or fragment thereof maybe free in solution, affixed to a solid support, recombinantly expressedon or chemically attached to a cell surface, or located intracellularly.The formation of binding complexes between NAAR polypeptide and thecompound being tested, may be measured by methods well known to thoseskilled in the art, such as the BIAcore (Upsala, Sweden). Anothertechnique provides for high throughput screening of compounds havingsuitable binding affinity to the protein of the invention as describedin published PCT application WO84/03564, and incorporated herein byreference in its entirety. Test substances that decrease NAAR expressionor activity are defined as inhibitors or antagonists of NAAR. Testsubstances that increase NAAR expression or activity are defined asactivators or agonists. Agents which modulate the expression or activityof the NAAR of the subject invention include, but are not limited toantisense oligonucleotides, ribozymes, and antibodies. These agents maybe made and used according to methods well known in the art.

In another embodiment of the invention, the NAAR polypeptide is used tobind FFAs, triglycerides, and cholesterol in vivo and remove thesemolecules from the bloodstream. This method comprises the step of:introducing an effective amount of NAAR polypeptide or fragment thereofto the bloodstream of an individual. In this embodiment, the NAARpolypeptide may further be expressed as a fusion protein with apolypeptide signal specifying excretion from the body. A preferredmethod of delivering NAAR polypeptides or biologically active fragmentsthereof to an individual includes direct, intravenous injection of saidpolypeptides or fragments in a physiologically acceptable solution(e.g., pH-buffered isotonic saline solutions, pH-buffered isotonicsaline solutions modified by addition of viscous elements such asglycerol). Alternatively, NAAR polynucleotides may be introduced toexpress NAAR polypeptides in the bloodstream. This method comprises thesteps of: i) constructing a recombinant viral vector corresponding to aportion of the genome of an adenovirus capable of infecting a celloperatively linked to the nucleotide sequence of the invention and aregulatory sequence directing its expression; ii) delivery of aneffective amount of the recombinant adenoviral vector to an individualwith or at risk of atherosclerosis, diabetes, or other lipid metabolismdisorders.

NAAR polypeptides, fragments thereof, or NAAR agonists may be used toprevent or treat atherosclerosis or arterial lipoprotein deposits ofFFAs, triglycerides, and cholesterol as determined by common medicaltechniques, such as determining the familial predisposition of theindividual for these disorders or actual analysis of the serumlipoprotein levels in the individual. Alternatively, NAAR polypeptides,fragments thereof, or NAAR agonists may be delivered to an individual atrisk of or suffering from diabetes or another lipid metabolism disorder.

In another embodiment, a NAAR polypeptide, fragment thereof, or NAARagonist is used in a physiologically acceptable composition to treatseptic shock or conditions associated with elevated serum levels oflipopolysaccharide (U.S. Pat. Nos. 5,932,536 and 5,948,756, whichdisclosures are hereby incorporated by reference in their entireties).

In another embodiment, a NAAR polypeptide, fragment thereof, or NAARagonist is applied to treatment and prevention of obesity, as NAARpeptides have appetite suppressive activities (U.S. Pat. No. 5,840,688,which disclosure is hereby incorporated by reference in its entirety).This method comprises the step of introducing a NAAR polypeptide,fragment thereof, or NAAR agonist in a physiologically acceptablesolution to an individual. Preferably, the individual is overweight orhas a body mass index (BMI) of 25 or greater.

Another embodiment of the subject invention provides compositions andmethods of selectively modulating the activity of the protein of theinvention. Modulation of NAAR activity would allow for the successfultreatment and management of liver-related and lipid metabolismassociated disorders.

Another embodiment of the invention relates to composition and methodsusing polynucleotide sequences encoding the protein of the invention orpart thereof to establish transgenic model animals (D. melanogaster, M.musculus), by any method familiar to those skilled in the art. Bymodulating in vivo the expression of the transgene with drugs ormodifier genes (activator or suppressor genes), animal models can bedeveloped that mimic human hormone-dependent disorders such as cancers.These animal models would thus allow the identification of potentialtherapeutic agents for treatment of the disorders. In addition,recombinant cell lines derived from these transgenic animals may be usedfor similar approaches ex vivo.

Protein of SEQ ID NO:30 (Internal Designation Clone 222588116-094-1-0-H2-F)

The cDNA of Clone 222588_(—)116-094-1-0-H2-F (SEQ ID NO:29) encodesNeurexinal protein of SEQ ID NO:30, comprising the amino acid sequence:MTSGSKCPSTDSGKEEYIATFKGSEYFCYDLSQNPIQSSSDEITLSFKTLQRNGLMLHTGKSADYVNLALKNGAVSLVINLGSGAFEALVEPVNGKFNDNAWHDVKVTRNLRQVTISVDGILTTTGYTQEDYTMLGSDDFFYVGGSPSTADLPGSPIQHESTFAEDPMFQSQTAQL. Accordingly, itwill be appreciated that all characteristics and uses of thepolypeptides of SEQ ID NO:30 described throughout the presentapplication also pertain to the polypeptides encoded by the human cDNAincluded in Clone 222588_(—)116-094-1-0-H2-F. In addition, it will beappreciated that all characteristics and uses of the polynucleotides ofSEQ ID NO:29 described throughout the present application also pertainto the nucleic acids comprising the human cDNA in Clone222588_(—)116-094-1-0-H2-F. A preferred embodiment of the invention isdirected toward the compositions of SEQ ID NO:29, SEQ ID NO:30 and Clone222588_(—)116-094-1-0-H2-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

The protein of SEQ ID NO:29, Neurexinal, is a splice variant of NeurexinI-alpha protein (Genbank accession number AB035356). The protein of theinvention displays a G-Laminin motif:FKTLQRNGLMLHTGKSADYVNLALKNGAVSLVINLGSGAFEALVEPVNGKFNDNAWHDVKVTRNLRQVTISVDGILTTTGYTQEDYTMLGSDDFFYVGGSPSTADLP (SEQ ID NO:81).Accordingly, some embodiments of the present invention comprise theamino acids of the G-Laminin motif and the polynucleotides encoding thesame.

Neurexins are neuronal cell-surface proteins. In addition to a functionas cell-adhesion molecules, neurexins serve as signaling receptors.Ligands for alpha-neurexins, called neuroxophilins, are similar topeptide hormones. Neurexins can also serve as receptors for latrotoxins.Neurexin splice variants contribute to target innervation and thetransition from neurite outgrowth to synaptogenesis or during synapticplasticity and regeneration. Neurexins are associated with mediatingneuronal processes and signaling exocytosis. Thus, these proteins play arole in neuronal development, neuronal abnormalities (e.g., seizures),and cancer.

Neurexinal, one neurexin isoform, is expressed in sympathetic neuronsduring target innervation and relative expression levels of splicevariants change during differentiation of individual neurons. Neurexinalis a membrane protein detectable only in neurons. Neurexinal expressionpeaks postnatally. Neurexinal plays a role as a cell adhesion moleculeand a signal transduction receptor.

Several ligands to Neurexinal, with putative functions in signaling andcell adhesion have been identified. Neurexinal binds to alpha-latrotoxinwith high affinity only in the presence of Ca2+. Alpha-latrotoxin is alarge peptide neurotoxin from black widow spider venom that causesmassive neurotransmitter release when applied to nerve terminals.Alpha-latrotoxin inserts into the membrane and forms a transmembrane ionpore after anchoring to either of its nerve terminal receptors. Inaddition, Neurexophilin binds tightly to Neurexinal and alpha-Neurexinbut not beta-Neurexins. Neurexophilin is synthesized at high levels onlyin inhibitory interneurons and functions as a signaling molecule.Dystroglycan is another physiological ligand for Neurexinal and mediatescell adhesion between brain cells. Dystroglycan, a cell surface protein,links the intracellular cytoskeleton to the extracellular matrix.Impairment of this linkage is instrumental in the pathogenesis ofmuscular dystrophies.

An embodiment of the invention is directed to a composition comprising anovel Neurexinal polypeptide sequence of SEQ ID NO:30.

A further embodiment of the invention is directed to a compositioncomprising a Neurexinal polypeptide fragment having biological activityof binding alpha-latrotoxin, neurexophilin, or dystroglycan.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:29 encoding aNeurexinal polypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a Neurexinal polypeptidefragment having biological activity of binding alpha-latrotoxin,neurexophilin, or dystroglycan.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a Neurexinal polypeptide or a biologicallyactive fragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing Neurexinalexpression. Preferably, the polynucleotides capable of directingNeurexinal expression are located in the 5′ regulatory region of theNeurexinal gene. Further preferably, these polynucleotides are locatedwithin 500 base pairs of the Neurexinal coding region. Thesepolynucleotides preferably comprise a promoter sequence. Techniquesknown in the art for introducing polynucleotide sequences to endogenoussequences are described in U.S. Pat. No. 5,641,670 and PCT WO962941,which disclosures are hereby incorporated by reference in theirentireties. Neurexinal protein produced by said host cell may be usedfor in vitro detection and purification methods as well as diagnosis andin vivo applications.

Neurexinal polypeptides bind alpha-latrotoxin, neurexophilin anddystroglycan. A preferred embodiment of the invention is a methodcomprising the step of: binding a Neurexinal polypeptide or fragmentthereof with an alpha-latrotoxin, neurexophilin or dystroglycan moleculeunder conditions that allow binding of Neurexinal polypeptide to saidmolecule. In a preferred embodiment, Neurexinal polypeptide is used topurify alpha-latrotoxin, neurexophilin or dystroglycan. In such method,Neurexinal polypeptide is preferably covalently or non-covalentlyattached to a solid matrix and allowed to bind alpha-latrotoxin,neurexophilin or dystroglycan using techniques well known in the art.This method comprises the steps of: i) washing the solid matrix to getrid of contaminants, ii) eluting the particle of interest using morestringent conditions. Additional aspects of this embodiment includemethods of using Neurexinal polypeptide to detect and quantifyalpha-latrotoxin, neurexophilin and dystroglycan using techniques commonin the art. This method comprises the steps of: i) obtaining abiological sample suspected of alpha-latrotoxin, neurexophilin anddystroglycan; ii) contacting said sample with a Neurexinal polypeptideor fragment thereof under conditions suitable for binding of Neurexinal;iii) and detecting the presence or absence of alpha-latrotoxin,neurexophilin and dystroglycan by detecting Neurexinal. Preferably,Neurexinal polypeptide or fragment thereof is covalently attached to adetectable compound. Alternatively, a detectable Neurexinal-specificantibody or fragment thereof may be used to detect Neurexinal. Thisembodiment is useful, for example, as a diagnostic tool for detectingneuronal levels of alpha-latrotoxin, neurexophilin and dystroglycan. Afurther aspect of the present invention involves the isolation,purification and characterization of Neurexinal. Specifically, thepresent invention extends to a novel neuronal receptor which is aregulator of neurotransmitter release, and thus mediatesalpha-latrotoxin toxicity in the presence of calcium.

An embodiment of the present invention relates to compositionscomprising Neurexinal polypeptides. The method of producing Neurexinalpolypeptides comprises the steps of: i) transfecting a mammalian hostcell with a recombinant expression vector comprising a polynucleotide ofthe present invention, and ii) purifying the produced protein.Neurexinal may be isolated according to any technique known in the artor disclosed herein. Preferably, an antibody directed against Neurexinalor a fragment thereof, is bound to a chromatographic support to form anaffinity chromatography column.

In another embodiment, the protein of the invention may be used totarget heterologous compounds (polypeptides or polynucleotides) to theneurons. For instance, a chimeric protein composed of Neurexinal or afragment thereof recombinantly or chemically fused to a compound,protein, or polynucleotide of interest would allow specific delivery tocells that express ligands for neurexinal, such as dystroglycan.Dystroglycan is expressed in neurons of the cerebral cortex,hippocampus, olfactory bulb, basal ganglia, thalamus, and hypothalamus.A method of targeting these cell populations would be valuable fortreatment of a number of neuronal and mood disorders such as epilepsy,stress disorder, schizophrenia, Huntingtons, Alzheimers and Parkinsonsdisease. In addition, the hypothalamus of obese individuals oftenexpresses lower than normal levels of leptin receptor. Thus,introduction of leptin receptor constructs or agonists to thehypothalamus with heterologous Neurexinal may be an effective treatmentfor obesity.

A preferred embodiment of the invention relates to compositions andmethods using the protein of the invention or fragment thereof to labelneurons in order to visualize any change in number, topology ormorphology. Such methods can be used to map neuronal tissue damageassociated with stroke; neurodegenerative conditions such asschizophrenia, Alzheimer's disease, epilepsy, stress disorder,Huntington's disease, and Parkinson's disease; and peripheralneuromuscular diseases such as myasthenia gravis and AIDS-relatedcomplex. For example, the protein of the invention or a fragment thereofmay be used to generate specific antibodies, which would in turn allowthe visualization of neurons, by methods well known to those skilled inthe art. Preferably, the antibody is labeled with any detectable moietyincluding, but not limited to, a fluorescent label, a radioactive atom,a paramagnetic ion, biotin, a chemiluminescent label or a label whichcan be detected through a secondary enzymatic or binding step. In asimilar fashion, antibodies raised against Neurexinal may be used toidentify particular cell types as Neurexinal is specifically expressedonly in neurons. Alternatively, quantitative analysis or detection ofthe protein of the invention, or of nucleic acids encoding Neurexinal,can be carried out by any other technique known to those skilled in theart. The invention further provides a method of diagnosing neurologicaldisease or conditions, including but not limited to those listed above.

In another embodiment, the invention relates to a method of usingcompounds that bind a Neurexinal polypeptide or fragment thereof. Suchmethod is directed toward diagnosis of disorders related to abnormalNeurexinal levels including neurodegenerative conditions such asschizophrenia, Alzheimer's disease, epilepsy, stress disorder,Huntington's disease, and Parkinson's disease. This method comprises thestep of contacting a compound that specifically binds Neurexinal with abiological sample. Preferred compounds include Neurexinal specificantibodies and antigen binding fragments thereof. Further preferredcompounds include those labeled with a detectable label, such as afluorescent, radioactive, or enzymatic tag. This method may rely onvarious diagnostic techniques, including immunoassays.

In another embodiment, the present invention includes an assay systemwhich may be prepared in the form of a test kit for the quantitativeanalysis of the extent of the presence of Neurexinal, or to identifycompounds that may mimic or block its activity. The system or test kitcomprises: a component that specifically binds Neurexinal andoptionally, a means for detecting said component. Preferred componentsare Neurexinal-specific antibodies and antigen-binding fragmentsthereof. Preferably, the antibody or fragment is detectably labeledwith, for example, a fluorescent, luminescent, or radioactive compound.Alternatively, the Neurexinal binding component may be labeled with adetectably enzymatic substrate and the optional means is the enzymerequired for detection.

It is a further object of the present invention to provide a method andassociated assay system for screening substances that are effective ineither mimicking the activity or combating the adverse effects ofNeurexinal in mammals.

A preferred embodiment of the invention is a method of screening fortest compounds that increase or decrease Neurexinal expression. Thismethod comprises the steps of: i) contacting a cell with a testsubstance, ii) detecting and quantifying Neurexinal expression levels,and iii) comparing Neurexinal expression in an exposed cell to that ofan unexposed control cell. Preferably, Neurexinal expression is studiedin neurons. Methods of detecting Neurexinal expression may be directedeither to Neurexinal transcripts (e.g., Northern blotting, in situhybridization, and other nucleotide detecting methods) or Neurexinalpolypeptides (e.g., Western blotting or immunohistological methods). Theexpression level of Neurexinal can be decreased in a biological sample,for example using antisense molecules.

A preferred embodiment of the invention is a method of screening forcompounds that increase or decrease Neurexinal activity. This methodcomprises the steps of: i) contacting a cell with a test substance, ii)detecting and quantifying Neurexinal activity levels, and iii) comparingNeurexinal activity in an exposed cell to that of an unexposed controlcell. Preferably, Neurexinal activity is detected by determining bindingof Neurexinal to alpha-latrotoxin, neurexophilin or dystroglycan.Binding may be tested by competitive binding assays withdetectably-labeled proteins from the above list.

Neurexinal activators or agonists are defined as compounds that increaseNeurexinal activity or expression. Neurexinal inhibitors or antagonistsare defined as compounds that decrease Neurexinal activity orexpression.

In yet a further embodiment, the invention is drawn to antagonists ofthe activity of Neurexinal. For example, the antagonist may be used tocounteract alpha-latrotoxin and used as an antivenom for black widowspider bites. A Neurexinal antagonist may further be used or to treatdisorders associated with abnormally high norepinephrine levels, such asPheochromocytoma, attention-deficit hyperactivity disorder (ADHD),idiopathic torsion dystonia, and monoamine oxidase A deficiency. Amethod of using a Neurexinal inhibitor comprises the step of:administering a Neurexinal inhibitor in a physiologically acceptablecomposition to an individual. Physiologically acceptable compositionsand carriers are discussed herein. Appropriate treatment will varydepending on the individual situation and may be determined by oneskilled in the art.

Neurexinal activators may be used to modulate and/or reverse thedegeneration of nerve terminals, to modulate synaptic transmission, orto treat other pathologies related to abnormally low levels ofNeurexinal expression. A method of using a Neurexinal activatorcomprises the step of: administering a Neurexinal activator in aphysiologically acceptably solution to an individual. Physiologicallyacceptable compositions and carriers are discussed herein. Appropriatetreatment will vary depending on the individual situation and may bedetermined by one skilled in the art.

Protein of SEQ ID NO:32 (Internal Designation Clone 119033105-066-4-0-F10-F)

The cDNA of Clone 119033_(—)105-066-4-0-F10-F (SEQ ID NO:31) encodesNPIASY protein of SEQ ID NO:32, comprising the amino acid sequence:MAAELVEAKNMVMSFRVSDLQMLLGFVGRSKSGLKHELVTRALQLVQFDCTPELFKKIKELYETRYAKKNSEPAPQPHRPLDPLTMHSTYDRAGAVPRTPLAGPNIDYPVLYGKYLNGLGRLPAKTLKPEVRLVKLPFFNMLDELLKPTELVPQNNEKLQESPCIFALTPRQVELIRNSRELQPGVKAVQVVLRICYSDTSCPQEDQYPPNIAVKVNHSYCSVPGYYPSNKPGVEPKRPCRPINLTHLMYLSSATNRITVTWGNYGKSYSVALYLVRQLTSSELLQRLKTIGVKHPELCKALVKEKLRLDPDSEIATTGVRVSLICPLVKMRLSVPCRAETCAHLQCFDAVFYLQMNEKKPTWMCPVCDKPAPYDQLIIDGLLSKILSECEDADEIEYLVDGSWCPIRAEKELSCSPQGAILVLGPSDANGLLPAPSVNGSGALGSTGGGGPVGSMENGKPGADVVDLTLDSSSSSEDEEEEEEEEEDEDEEGPRPKRRCPFQKGLVPAC. Accordingly, it will be appreciated that allcharacteristics and uses of the polypeptides of SEQ ID NO:32 describedthroughout the present application also pertain to the polypeptidesencoded by the human cDNA included in Clone 119033_(—)105-066-4-0-F10-F.In addition, it will be appreciated that all characteristics and uses ofthe polynucleotides of SEQ ID NO:31 described throughout the presentapplication also pertain to the nucleic acids comprising the human cDNAin Clone 119033_(—)105-066-4-0-FI O—F. A preferred embodiment of theinvention is directed toward the compositions of SEQ ID NO:31, SEQ IDNO:32 and Clone 119033_(—)105-066-4-0-F10-F. Also preferred arepolypeptide fragments having a biological activity as described hereinand the polynucleotides encoding the fragments.

The protein of SEQ ID NO:32, NPIASY, is a novel polymorphic variant ofProtein Inhibitor of Activated STAT Protein (PIASY) (accession numbers075926). The protein of the invention displays a SAP domain:VMSFRVSDLQMLLGFVGRSKSGLKHELVTRALQLV (SEQ ID NO:82). In addition, NPIASYdisplays a MIZ Zinc finger motif:VSLICPLVKMRLSVPCRAETCAHLQCFDAVFYLQMNEKETCAHLQCFDAVFYLQMNEK (SEQ IDNO:83).

Activation of early response genes by interferons (IFNs) and othercytokines requires tyrosine phosphorylation of a family of transcriptionfactors termed signal transducer and activator of transcripton (STAT)proteins. STAT proteins relay signals from activated cell surfacereceptors directly to the nucleus and play a critical role in geneinduction by a variety of hematopoietic cytokines and hormones, such asthe interleukin 6 (IL6) family of cytokines, epidermal growth factor,and leptin. All STAT proteins contain a DNA biding domain, a Srchomology 2 domain and a transactivation domain necessary fortranscriptional activation of target gene expression. Janus kinases(JAK) are cytoplasmic protein kinases which, upon cytokine signaling,phosphorylate STAT proteins on a specific tyrosine residue. Tyrosinephosphorylated STAT proteins dimerize through specific reciprocalSH2-phosphotyrosine interactions and translocate from the cytoplasm tothe nucleus. Once in the nucleus, STAT proteins stimulate thetranscription of specific target genes by binding to response elementsin their promoters.

The binding of proteins from the Protein Inhibitor of Activated STAT(PAIS) family inhibits the STAT signaling pathway. The PIAS-STATinteraction depends on the tyrosine phosphorylation of STAT proteins.

NPIASY is localized in the nucleus. Upon Interferon stimulation, NPIASYbecomes associated with STAT1. An LXXLL coregulator sequence at theNH2-terminus of NPIASY, although not involved in NPIASY-STAT1interaction, is required for repression of STAT1-mediated geneactivation. NPIASY is a transcriptional corepressor of STAT1. Also,NPIASY acts as a potent inhibitor of transcriptional activity ofandrogen receptor in prostate cancer cells. NPIASY binds to androgenreceptor but does not affect the DNA biding activity of androgenreceptor. The NH2 terminal LXXLL signature motif of NPIASY, although notrequired for NPIASY-androgen receptor interaction, is essential for theinhibitory activity of NPIASY. In addition, NPIASY binds to p53 andinhibits p53-mediated transactivation. However, NPIASY does not affectp53-mediated apoptosis. NPIASY regulates p53-mediated functions anddirects p53 into a transactivation-independent mode of apoptosis.

An embodiment of the invention is directed to a composition comprising aNPIASY polypeptide sequence of SEQ ID NO:32.

A further embodiment of the invention is directed to a compositioncomprising a NPIASY polypeptide fragment having a biological activity ofbinding to STAT, p53, or Androgen Receptor.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:31 encoding a NPIASYpolypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a NPIASY fragment having abiological activity of binding to STAT, p53, or Androgen Receptor.

NPIASY polypeptides bind to STAT, androgen receptor, and p53, which areoften associated with cancers including T cell leukemias, breast,prostate, colorectal, pancreatic, lung, and esophageal cancers. Apreferred embodiment of the invention is a method comprising the step ofbinding a NPIASY polypeptide, or fragment thereof with STAT, androgenreceptor or p53 molecules under conditions that allow binding of NPIASYpolypeptide to said molecules. This method may be directed to in vitropurification and detection methods as well as in vivo methods to inhibitSTAT, p53, or Androgen Receptor. Such methods are discussed herein.

In a preferred embodiment, NPIASY polypeptide is used to purify STAT,androgen receptor or p53. In such method, NPIASY polypeptide ispreferably covalently or non-covalently attached to a solid matrix andallowed to bind STAT, androgen receptor or p53 using techniques wellknown in the art. This method comprises the steps of: i) washing thesolid matrix to get rid of contaminants, ii) eluting the particle ofinterest using more stringent conditions. Additional aspects of thisembodiment include methods of using NPIASY polypeptide to detect andquantify STAT, androgen receptor, or p53 using techniques common in theart. This method comprises the step of: i) obtaining a biological samplesuspected of containing STAT, androgen receptor, or p53; ii) contactingsaid sample with a NPIASY polypeptide or fragment thereof underconditions suitable for binding of NPIASY; and iii) detecting thepresence of STAT, androgen receptor, or p53 by detecting NPIASY.Preferably, NPIASY polypeptide or fragment thereof is covalentlyattached to a detectable compound. Alternatively, a detectableNPIASY-specific antibody or fragment thereof may be used to detectNPIASY. This embodiment is useful, for example, as a diagnostic tool fordetecting risk of p53, STAT, or Androgen Receptor related cancers suchas T cell leukemias, breast, prostate, colorectal, pancreatic, lung, andesophageal cancers. In addition, this method may be used to diagnosedisorders associated with a low level of the above proteins includingmuscle atrophy and sexual development defects.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a NPIASY polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing NPIASY expression.Preferably, the polynucleotides capable of directing NPIASY expressionare located in the 5′ regulatory region of the NPIASY gene. Furtherpreferably, these polynucleotides are located within 500 base pairs ofthe LIRION coding region. These polynucleotides preferably comprise apromoter sequence. Techniques known in the art for introducingpolynucleotide sequences to endogenous sequences are described in U.S.Pat. No. 5,641,670 and PCT WO9629411, which disclosures are herebyincorporated by reference in their entireties. NPIASY protein purifiedfrom said host cell may be used for in vitro methods of detection andpurification, as well as diagnostic and in vivo applications.

An embodiment of the present invention relates to a method of producingcompositions comprising NPIASY polypeptides. The method of producingNPIASY polypeptides comprises the steps of: i) transfecting a host cellwith a recombinant expression vector comprising a polynucleotide of thepresent invention, and ii) purifying the produced protein. NPIASY may bepurified by any technique known in the art or disclosed herein.Preferably, an antibody directed against NPIASY or part thereof,preferably, an antibody directed against the C-terminal sequence ofNPIASY polypeptide, may be bound to a chromatographic support to form anaffinity chromatography column. Alternatively, NPIASY polypeptides maybe produced by a method comprising the step of: i) transfecting a hostcell with a polynucleotide capable of directing NPIASY expression.Preferably, the polynucleotides capable of directing NPIASY expressionare located in the 5′ regulatory region of the NPIASY gene. Furtherpreferably, these polynucleotides are located within 500 base pairs ofthe NPIASY coding region. These polynucleotides preferably comprise apromoter sequence, as discussed above.

In another embodiment, the invention is directed to a method ofdetecting NPIASY polypeptide in a biological sample, said methodcomprising the steps of: i) contacting a biological sample with anantibody or antibody fragment that specifically binds NPIASYpolypeptide; and ii) detecting the antigen-antibody complex formed. Theantibody or antibody fragment may be monoclonal or polyclonal. Inaddition, the antibody or antibody fragment may be primarily orsecondarily labeled by a detectable compound common in the art (e.g.,radioactive, fluorescent, luminescent, or enzymatic). This method may beapplied to diagnosis of NPIASY-related disorders. For example, anabnormally low level of NPIASY may indicate a high risk of p53, STAT, orAndrogen Receptor related cancers such as T cell leukemias, breast,prostate, colorectal, pancreatic, lung, and esophageal cancers. Anabnormally high level of NPIASY may indicate a risk of growth and cellproliferation defects associated with the above proteins such asincomplete sexual development and muscle atrophy.

In some embodiments, the invention also concerns a diagnostic kit fordetecting the presence of NPIASY polypeptide. This kit comprises: i) apolyclonal or monoclonal antibody or fragment thereof that specificallybinds a NPISAY polypeptide; and optionally, ii) a reagent allowing thedetection of the antigen-antibody complexes formed. Preferably, theantibody, or antibody fragment is detectably labeled. Such labelsinclude fluorescent, luminescent, and radioactive compounds, as well asenzymatic substrates. The optional reagent may provide a detectablesignal that either binds to the antibody or reacts with the label on theantibody. NPIASY antibodies may be used to diagnose any of the disorderslisted above. To diagnose such disorders, an appropriate biologicalsample can be tested to determine the level of NPIASY being produced.

In another embodiment, the invention provides methods for regulating anIFN-associated immune response mediated by STAT. The immune responseincludes an anti-viral or anti-tumor response mediated by IFN secretedby B and/or T cells. This method comprises the step of introducing apolynucleotide construct comprising polynucleotides encoding a NPIASYpolypeptide to an IFN-responsive cell. Preferrably, the polynucleotideconstruct comprises an expression control element operably linked to theNPIASY-encoding polynucleotide. Suitable compositions of polypeptides orpolynucleotides of the present invention are useful as a method oftreatment of IFN-related pathologies such as inflammation.

An embodiment of the invention provides for a method of screening testsubstances for modulators of NPIASY expression. This method comprisesthe steps of: i) contacting a cell with a test substance; ii) detectingand quantifying NPIASY expression; and ii) comparing NPIASY expressionin the cell after exposure to the test substance to that of an unexposedcontrol cell. NPIASY expression is determined by methods common to theart or included herein, by detecting NPIASY polynucleotides orpolypeptides. An example of this method comprises the steps of: i)culturing two equivalent cell samples; ii) adding a test substance toone of the cultures and not the other; iii) harvesting both cultures ata specified time; iv) purifying the mRNA from each sample of cells; v)comparing the level of NPIASY mRNA in each sample by Northern blot,RTPCR, or another method common to the art. The invention provides fordesign and use of specific polynucleotide probes and primers, asdiscussed herein. An additional example comprises the steps of: i)having two equivalent cultures of cells; ii) adding a test substance toone of the cultures and not the other; iii) harvesting both cultures;iv) purifying the protein from each sample of cells; v) comparing thelevel of NPIASY polypeptides in each sample by Western blot,immunohistochemistry, or another method common to the art. The inventionprovides for design and use of specific antibodies and antibodyfragments, as discussed herein.

In another embodiment, a NPIASY polypeptide or a fragment thereof, maybe used to screen for compounds that activate or inhibit NPIASYactivity. This method comprises the steps of: i) contacting a NPIASYpolypeptide or fragment thereof with a test substance and ii) monitoringNPIASY activity. NPIASY binds to STAT, androgen receptor, and p53. Thus,NPIASY activity may be monitored upon addition of the test substance bycompetitive binding assays with either STAT, androgen receptor, or p53.In this aspect of the invention, a NPIASY polypeptide or fragmentthereof may be free in solution, affixed to a solid support,recombinantly expressed on or chemically attached to a cell surface, orlocated intracellularly. The formation of binding complexes betweenNPIASY polypeptide and the compound being tested, may be measured bymethods well known to those skilled in the art, such as the BIAcore(Upsala, Sweden). Another technique provides for high throughputscreening of compounds having suitable binding affinity to the proteinof the invention as described in published PCT application WO84/03564,and incorporated herein by reference in its entirety. Test substancesthat decrease NPIASY expression or activity are defined as inhibitors orantagonists of NPIASY. Test substances that increase NPIASY expressionor activity are defined as activators or agonists. Antagonists of thepolypeptides or polynucleotides of the present invention are used tostimulate, promote, or facilitate progression through the cell cycle,such as in the cellular regeneration of terminally differentiatedcardiac myocytes or tissues, e.g., striated muscle myocytes. Forexample, this could allow restoration of damaged myocardium aftercardiac injury, myocardial infarction, myocarditis, cardiomyopathy,trauma, as a consequence of cardial surgery, etc., or repletion ofstriated muscle exhausted by muscular dystrophy. Agonists ofpolypeptides or polynucleotides of the present invention are useful intreatment of pathologies such as but not limited to hyperproliferativediseases such as cancer (e.g., leukemia, lymphoma, breast cancer, coloncancer, prostate cancer) coronary artery disease, pulmonary vascular,obstructive disease, either primary or as a feature of Eisenmenger'ssyndrome, and other disorders of abnormal cellular proliferation. Agentswhich modulate the expression or activity of NPIASY include, but are notlimited to antisense oligonucleotides, ribozymes, and antibodies. Theseagents may be made and used according to methods well known in the art.

In a further embodiment, expression of the polypeptides encoded by thenucleic acids is expected to prevent, ameliorate, or lessen the cellcycle defect of the host cell, or to restore normal cell cycleprogression of the host cell. Whether provided via nucleic acid orpolypeptides delivered directly to cells, the therapeutic formulationsof the invention can also be used as adjuncts to other forms of therapy,including but not limited to chemotherapy, and radiation therapy.

In another preferred embodiment, the protein of the invention can beused to modulate and/or characterize fertility, including for thetreatment or diagnosis of infertility, and for contraception. NPIASYinactivates Androgen receptor which is required for the activation ofgenes essential for spermatogenesis. Thus, over-expression or activationof NPIASY can be used to repress Androgen receptor transcriptionactivity and thereby inhibit fertility. For example, for contraception,the expression of genes for spermatogenesis can be artificiallydisrupted, for example by increasing the protein level usingpolynucleotides encoding the protein, using the protein itself, or usingactivators of protein expression or activity. Alternatively, forinfertility, the protein level can be decreased using inhibitors such asantisense oligonucleotides, antibodies, dominant negative forms of theprotein, and using heterologous compounds that inhibit proteinexpression or activity. Similarly, the cause of infertility in manypatients can be detected by detecting the level of expression of thepresent protein, where an abnormal level of activity or expression ofthe protein indicates that a cause of infertility involves the NPIASYrepression. Such a diagnosis would also point to methods of treating theinfertility, e.g. by increasing or decreasing the expression oractivation of the present protein.

Another embodiment of the invention relates to composition and methodsusing polynucleotide sequences encoding the protein of the invention ora fragment thereof to establish transgenic model animals (D.melanogaster, M. musculus), by any method familiar to those skilled inthe art. By modulating in vivo the expression of the transgene withdrugs or modifier genes (activator or suppressor genes), animal modelscan be developed that mimic human hormone-dependent disorders such ascancers. These animal models would thus allow the identification ofpotential therapeutic agents for treatment of the disorders. Inaddition, recombinant cell lines derived from these transgenic animalsmay be used for similar approaches ex vivo.

Protein of SEQ ID NO:34 (Internal Designation 125402 105-074-4-0-F3-F)

The cDNA of 125402_(—)105-074-4-0-F3-F (SEQ ID NO:33) encodes proteinvCRTL-1 of SEQ ID NO:34, comprising the amino acid sequence:MLLLSLTLSLVLLGSSWGCGIPAIKPALSFSQRIVNGENAVLGSWPWQVSLQDSSDFHFCGGSLISQSWVVTAAHCNVSPGRHFVVLGEYDRSSNAEPLQVLSVSRAITHPSWNSTTMNNDVTLLKLASPAQYTTRISPVCLASSNEALTEGLTCVTTGWGRLSGVGNVTPARLQQVALPLVTVNQCRQYWGSSITDSMICAGGAGASSCQGDSGGPLVCQKGNTWVLIGIVSWGTKNCNVRAPAVYTRVSKFSTWINQVIAYN. Accordingly, it will be appreciated that allcharacteristics and uses of polypeptides of SEQ ID NO:34 describedthroughout the present application also pertain to the polypeptidesencoded by the human cDNA included in 125402_(—)105-074-4-0-F3-F. Inaddition, it will be appreciated that all characteristics and uses ofthe polynucleotides of SEQ ID NO:33 described throughout the presentapplication also pertain to the nucleic acids comprising the human cDNAin 125402_(—)105-074-4-0-F3-F. A preferred embodiment of the inventionis directed toward the compositions of SEQ ID NO:33, SEQ ID NO:34, and125402_(—)105-074-4-0-F3-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

The cDNA of SEQ ID NO:33 is a novel polymorphic variant of the humanchymotrypsin-like protease CTRL-1 encoded by a gene located onchromosome 16, specifically at position 16q22.1. The 264 amino-acidprotein of vCRTL-1 contains a signal peptide (MLLLSLTLSLVLLGSSWG) (SEQID NO:84) and a serine protease domain and belongs to the serineprotease family.

Proteases are key components in a broad range of biological pathways andcan be classified into four groups according to their catalyticmechanisms: the serine, cysteine (thiol), aspartic (carboxyl), andmetalloproteases. Chymotrypsins are members of a family of enzymes knownas serine proteases, so named because they utilize an activated serineresidue in their substrate-binding site to catalyze the hydrolysis ofcertain peptide bonds. The pancreatic subfamily of proteases comprisestrypsins, chymotrypsins, kallikrein, and elastases. Chymotrypsins arethe most abundant among the pancreatic proteases and may represent10-20% of the total protein synthesized by the exocrine pancreas.vCRTL-1 is a digestive enzyme expressed and synthesized by pancreaticacinar cells as an inactive zymogen precursor that must be cleaved toform an active protease. vCRTL-1 displays chymotrypsin- andelastase-2-like activities and hydrolyzes the amide bonds of substrateshaving tyrosine, phenylalanine, or leucine residues at the P1 position,and preferably a proline in the P2 position. The secretion of vCRTL-1can be induced by the presence of protease inhibitors in the pancreas orintestinal fluid (in particular duodenal fluid).

An embodiment of the invention is directed to a composition comprising avCRTL-1 polypeptide sequence of SEQ ID NO:34.

A further embodiment of the invention is directed to a compositioncomprising a vCRTL-1 polypeptide fragment having a biological activityof protein substrate binding or serine protease activity.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:33 encoding a vCRTL-1polypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a vCRTL-1 polypeptidefragment having a biological activity of protein substrate binding orserine protease activity.

A further embodiment of the invention is directed to a compositioncomprising an antibody directed against the vCRTL-1 polypeptide sequenceof SEQ ID NO: 34 or a vCRTL-1 polypeptide fragment having a biologicalactivity of protein substrate binding or serine protease activity.Preferably, the antibody specifically binds to vCRTL-1 but not toCTRL-1. Even more preferably, the antibody recognizes the SLQDSSDFHF(SEQ ID NO:85) amino-acid sequence.

An additional preferred embodiment of the invention is a method ofbinding vCRTL-1 polypeptides with a vCRTL-1-specific antibody orvCRTL-1-binding fragment thereof. This method comprises the step ofcontacting a vCRTL-1 polypeptide with a vCRTL-1-binding antibody orvCRTL-1-binding fragment thereof under conditions that allow binding.This method may be applied to detection and purification of vCRTL-1.These aspects are discussed in detail herein.

Another embodiment relates to a method of producing a recombinantvCRTL-1 polypeptide comprising the steps of: i) transfecting a host cellwith an appropriate expression vector comprising a polynucleotideencoding vCRTL-1 and ii) purifying the produced protein. The host cellsare cultured under conditions whereby the nucleic acid sequence codingfor this particular protein is expressed. After a suitable amount oftime for the product to accumulate, the protein is purified from thehost cells or medium surrounding the cells. Introduction of anexpression vector incorporating a nucleic acid sequence coding forvCRTL-1 into a host cell can be performed in a variety of ways, such asbut not limited to calcium or lithium chloride treatment,electroporation, and lipofection. Also in this embodiment, a milk animalcan be used to produce vCRTL-1 in the milk, thereby generating asignificant amount of this particular protein after purification. Anytype of animal that produces enough milk can be used in this aim suchas, but not limited to, sheep, goat, and cow. These animals can begenerated with any method of targeting overexpression of vCRTL-1 in themilk. The protein of the invention may be purified using any techniquewell-known to those skilled in the art including those disclosed in theU.S. Pat. No. 6,268,487, which disclosure is hereby incorporated byreference in its entirety. Preferably, an antibody or antigen-bindingfragment directed against vCRTL-1 or a fragment thereof may be bound toa chromatographic support to form an affinity chromatography column.Even more preferably, the antibody binds specifically to vCRTL-1 but notto CTRL-1.

A preferred embodiment of the invention is a method of using vCRTL-1polypeptide or a biologically active fragment thereof to proteolyticallycleave a substrate protein. This method comprises the step of contactingvCRTL-1 with a protein substrate under conditions that allow vCRTL-1protease activity. This method is directed to both in vitro and in vivouses of the protein that include biochemical applications and preventionand treatment of vCRTL-1-related disorders such as chronic and acutepancreatits and cystic fibrosis.

In another embodiment, the present invention is to provide a method ofeffectively inducing vCRTL-1 proteolytic activity to digest a wide rangeof proteins in a biological sample. This method can be performed eitherin vitro or in vivo and comprises the step of contacting an effectiveamount of vCRTL-1 polypeptide or a biologically active fragment thereofwith a biological sample under conditions that allow vCRTL-1 proteolyticactivity. For example, such a method can be used in vitro for removingproteins in a DNA preparation or for removing enzymes after anyenzymatic reaction. Such a composition can be used alone or as a“cocktail” with other proteases. Such protease cocktails are widely usedin assays to degrade proteins susceptible of interfering with an assayto be performed.

In another embodiment, vCRTL-1 or fragment thereof can be used incombination with a detergent for the removal of stains having a proteincomponent, similar to the use of proteases described in U.S. Pat. No.5,599,400, which disclosure is incorporated by reference in itsentirety. The composition can contain known detergent constituents suchas surfactants, foam enhancers and fillers. vCRTL-1 polypeptides can beincluded in a detergent composition or can be combined with otherconstituents at the time of use as an additive. The detergent additivecan be formulated as a liquid, powder, granulate or slurry.

A further embodiment of the present invention is directed to substancesthat increase or decrease vCRTL-1 expression, and to a method ofscreening for such substances comprising the steps of: i) contacting acell with a test substance, ii) quantifying and detecting vCRTL-1expression levels, and iii) comparing vCRTL-1 expression in an exposedcell to that of an unexposed control cell. vCRTL-1 expression may bedetermined by any specific nucleotide or protein detection techniquescommon to the art (e.g., Northern or Western blotting) Preferably,vCRTL-1 expression is studied in pancreatic cells. The expression levelof vCRTL-1 can be decreased in a biological sample, for example usingantisense molecules.

A further embodiment of the present invention is directed to substancesthat increase or decrease vCRTL-1 activity, and to a method of screeningfor such substances comprising the steps of: i) contacting a cell with atest substance and ii) detecting vCRTL-1 activity, and iii) comparingvCRTL-1 proteolytic activity in an exposed cell to that of an unexposedcontrol cell. Preferably, vCRTL-1 expression is studied in pancreaticcells. The activity of vCRTL-1 may be inhibited using direct or indirectinhibitor molecules or antagonistic antibodies.

Activators or agonists of vCRTL-1 are substances that increase theactivity or expression of vCRTL-1. Inhibitors or antagonists of vCRTL-1are substances that decrease the activity or expression of vCRTL-1.

The present invention also provides animal models generated bymodulating the expression or activity of the present protein in one ormore tissues of the animal and preferably in pancreas. Such animals areuseful for a number of purposes, because they represent an in vivo assaymethod for testing candidate molecules potentially useful for thetreatment of various pathophysiological aspects of diseases specificallyrelated to the activity of vCRTL-1 such as acute or chronicpancreatitis. Study of the phenotype of such models can also allow theidentification of additional human equivalent diseases caused by orlinked with vCRTL-1 deficiency. These animals can be generated with anymethod of targeting overexpression or inactivation of vCRTL-1. Suchmodels are extremely useful, e.g. in the assessment of candidatetherapies and drugs for the treatment of such diseases and conditions.

In another embodiment, the current invention is used to diagnosediseases or disorders associated with abnormal vCRTL activity. Inparticular, it is useful in diagnosing patients with exocrine pancreaticinsufficiency which results in a dramatic reduction of vCRTL-1 activity.Examples of such diseases and disorders include, but are not limited to,chronic or acute pancreatits and cystic fibrosis. In addition, vCRTL isassociated with metastatic tumor activity, in particular, that ofpancreatic tumors. Thus, higher than normal levels of vCRTL may beindicative of metastatic activity. The method includes the steps of: i)contacting a fluid or tissue sample with a compound capable ofselectively binding vCRTL-1 polypeptide or polynucleotide and ii)detecting the level of vCRTL-1 polypeptide or polynucleotide in thesample. Preferably, a difference in the level or other property in thesample relative to in a control sample indicates the presence of thedisorder or a propensity for developing disorder. Preferably, samples tobe analyzed include, but are not limited to, intestinal fluids and inparticular duodenal juice, homogenates of pancreatic tissue, urine andplasma. More preferably, the fluid or tissue sample is obtained from anindividual suspected of suffering from the disorder, or at risk ofdeveloping the disorder. For example, a polyclonal or monoclonalantibody or any antigen binding active fragment thereof or a nucleicacid probe may be used. Preferably, the antibodies used in thisembodiment are specifically directed against the variant vCRTL-1polypeptides and do not recognize CRTL-1 polypeptides. Detection can becarried out directly or indirectly with known immunohistological orimmunofluorescent processes. vCRTL-1 binding molecules may be directlylabeled with generally known compounds, including, but not limited to,enzymes such as alkaline phosphatase and peroxidase and fluorescent dyessuch as FITC, rhodamine, and Texas-Red. However, labeling can also occurindirectly by using secondary reagents. Also in this embodiment,diagnosis may be facilitated by the identification of the variantvCRTL-1 using well known PCR or RT-PCR techniques and in particular inwith “real-time” PCR system. Alternatively, using such a method, thepresent invention provides a tool to correlate modulations in theexpression of vCRTL-1 with certain pathologies. Thus, the presentinvention provides a novel candidate gene for such conditions. In afurther embodiment, the invention includes a test kit useful for thequantification of the amount of vCRTL-1 in a biological sample. Thelevel of vCRTL-1 polypeptide is useful in evaluating the degree ofseverity of pancreatic disorders that have been clinically ascertainedand also represents a useful marker in examining the pathophysiology andpossible treatment modalities in the animal model of acute pancreatitis.In addition, vCRTL-1 level may be useful in the estimation of tumorinvasiveness and metastasis ability, preferably in the case ofpancreatic cancers. In this embodiment, the application of such assayscan be used to monitor the progress of therapy administered to treatthese or other conditions. Further, the assays can be used as a measureof toxicity, for example, during clinical testing of new drugs to assessthe impact on tissue degradation. Thus, the assays may be applied in anysituation wherein vCRTL-1 may be used as an index of the condition,treatment, or effect of substances directly administered to the subject.Thus, the condition of a patient can be monitored continuously and thequantified amount of vCRTL-1 in the pathological sample can be comparedwith the amount quantified in a biological sample of a normal individualor with the previous analysis of the same patient. In this embodiment,this marker can be measured effectively in intestinal fluids and inparticular in duodenal juice, homogenates of pancreatic tissue, urineand plasma, by any suitable method, including immunoassays. The kitcomprises at least one immunological binding partner (e.g., a monoclonalor polyclonal antibody specific for vCRTL-1) and optionally, a secondarydetectable marker. Preferably, the antibody or antigen-binding fragmentthereof used in the kit is specifically directed against the variantvCRTL-1 polypeptides and not to CTRL-1 polypeptides.

A further embodiment of the present invention is to provide novelmethods and compositions useful for treating individuals at risk ofdeveloping or suffering from a disorder associated with insufficientsecretion of vCRTL-1. These methods and compositions are useful for thetreatment of exocrine pancreatic deficiency characterized bysteatorrhoea such as chronic or acute pancreatits and cystic fibrosis.This method comprises the step of: administering an effective amount ofvCRTL-1 polypeptide, a biologically active fragment thereof, or agonistto an individual with or at risk of one of the listed conditions.Preferably, vCRTL-1 or agonist compositions are delivered in combinationwith a physiologically acceptable carrier, such as a saline solution orother physiologically buffer. The particular amount of the compositionsof the invention that will be administered to the individual for anyparticular condition will depend on the clinical condition of thepatient, and other factors such as the weight, age, and route ofdelivery. Such compositions may be administered by any suitable routeincluding, but not limited to, oral, aerosol, intravenous,intramuscular, intraperitoneal, or subcutaneous routes. Thesecompositions can comprise the protein of the invention, and, optionally,one or more other types of protease, or any other compound of interest.This co-administration may be by simultaneous administration or byseparate or sequential administrations. All of these additionalcomponents may be either obtained from natural sources or produced byrecombinant genetic engineering techniques and/or chemical modification.

A further embodiment of the present invention is to provide novelmethods and compositions useful for treating individuals at risk ofdeveloping or suffering from a disorder associated with higher thannormal levels of vCRTL-1. This method is useful for the treatment ofmetastatic cell growth, in particular, pancreatic cancers. This methodcomprises the step of administering an effective amount of a vCRTL-1antagonist to an individual with or at risk of developing a metastaticcell growth. Preferably, vCRTL-1 antagonist compositions are deliveredin combination with a physiologically acceptable carrier, such as asaline solution or other physiologically buffer. The particular amountof the compositions of the invention that will be administered to theindividual for any particular condition will depend on the clinicalcondition of the patient, and other factors such as the weight, age, androute of delivery. Such compositions may be administered by any suitableroute including, but not limited to, oral, aerosol, intravenous,intramuscular, intraperitoneal, or subcutaneous routes.

Protein of SEQ ID NO:36 (Internal Designation Clone 107640105-036-2-0-3-F)

The cDNA of Clone 107640 105-036-2-0-H3-F (SEQ ID NO:35) encodes the 447amino acid LIRION protein of SEQ ID NO:36 comprising the amino acidsequence: MIPTFTALLCLGLSLGPRTHMQAGPLPKPTLWAEPGSVISWGNSVTIWCQGTLEAREYRLDKEESPAPWDRQNPLEPKNKARFSIPSMTEDYAGRYRCYYRSPVGWSQPSDPLELVMTGAYSKPTLSALPSPLVTSEKSVTLLCQSRSPMDTFLLIKERAAHPLLHLRSEHGAQQHQAEFPMSPVTSVHGGTYRCFSSHGFSHYLLSHPSDPLELIVSGSLEDPRPSPTRSVSTAAGPEDQPLMPTGSVPHSGLRRHWEVLIGVLVVSILLLSLLLFLLLQHWRQGKHRTLAQRQADFQRPPGAAEPEPKDGGLQRRSSPAADVQGENFCAAVKDTQPEDGVEMDTRSPHDEDPQAVTYAKVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSFTLRQKATEPPPSQEGASPAEPSVYATLAIH. Accordingly, it will be appreciated that allcharacteristics and uses of polypeptides of SEQ ID NO:36 describedthroughout the present application also pertain to the polypeptidesencoded by the human cDNA included in Clone 107640 105-036-2-0-H3-F. Inaddition, it will be appreciated that all characteristics and uses ofthe polynucleotides of SEQ ID NO:35 described throughout the presentapplication also pertain to the nucleic acids comprising the human cDNAin 107640 105-036-2-0-H3-F. Also preferred are fragments having abiological activity as described therein and the polynucleotidesencoding the fragments.

LIRION, the protein of SEQ ID NO:36, represents a novel variant form ofthe ILT-3 receptor precursor (Genbank entry U82979) starting with a 259amino acid long extracellular domain including a peptide signal sequence(MIPTFTALLCLGLSLGPRTHMQA) (SEQ ID NO:86) and two C2 typeimmunoglobulin-like-domains, followed by a transmembrane region(VLIGVLVVSILLLSLLLFLLL) (SEQ ID NO:87) and a 167 amino acid longintracellular domain which contains three Immunoreceptor Tyrosine-basedInhibitory Motifs (ITIMs).

Immune system cellular activity is controlled by a complex network ofcell surface interactions and associated signaling processes. Uponreceptor binding, cellular activity is regulated by a balance betweenactivating and inhibitory signals. Many receptors that mediate positivesignaling have cytoplasmic tails containing sites of tyrosinephosphorylation known as Immunoreceptor Tyrosine-based Activation Motifs(ITAMs). The inhibitory pathways involve receptors having ITIMs, whichprovide binding sites for protein tyrosine phosphatases.

The cytolytic activity of Natural Killer (NK) cells is regulated by abalance between positive signals that initiate cell function andinhibitory signals which prevent the cell activity. Normal cells areprotected from NK cells by cell surface expression of MHC class Iproteins that are recognized by inhibitory receptors on the NK cells.These receptors, known as Killer Inhibitory Receptors (KIRs) send anegative signal to the cell upon engagement and down regulate NK cellcytotoxic activity. MHC class I expression is often down-regulated invirally infected and in tumor cells, which renders these cellssusceptible to NK cell attack. The KIRs are immunoglobulin superfamilyreceptors with two ITIMs. These phosphorylated ITIMs recruit tyrosinephosphatases which dephosphorylate molecules in the signal transductionpathway and prevent cell activation.

LIRION, the protein of the invention, is an inhibitoryImmunoglobulin-Like Transcript (ILT) receptor. This class of receptor isclosely related to the KIRs and functions in the same manner. LIRION isselectively expressed on myeloid antigen presenting cells (APCs), i.e.dendritic cells, monocytes and macrophages and negatively regulates thefunctional response of APCs triggered by stimulatory receptors. Inaddition to its inhibitory function, LIRION is involved in antigenuptake and processing.

Preferred LIRION polypeptides for use in the methods described belowinclude the polypeptides comprising the amino sequence of:GPLPKPTLWAEPGSVISWGNSVTIWCQGTLEAREYRLDKEESPAPWDRQNPLEPKNKARFSIPSMTEDYAGRYRCYYRSPVGWSQPSDPLELVMTGAYSKPTLSALPSPLVTSEKSVTLLCQSRSPMDTFLLIKERAAHPLLHLRSEHGAQQHQAEFPMSPVTSVHGGTYRCFSSHGFSHYLLSHPSDPLELIVSGSLEDPRPSPTRSVSTAAGPEDQPLMPTGSVPHSGLRRHWEVLIGVLVVSILLLSLLLFLLLQHWRQGKHRTLAQRQADFQRPPGAAEPEPKDGGLQRRSSPAADVQGENFCAAVKDTQPEDGVEMDTRSPHDEDPQAVTYAKVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSFTLRQKATEPPPSQEGASPAEPSVYATLAIH (SEQ ID NO:88);

A polypeptide comprising the amino acid sequence of:GPLPKPTLWAEPGSVISWGNSVTIWCQGTLEAREYRLDKEESPAPWDRQNPLEPKNKARFSIPSMTEDYAGRYRCYYRSPVGWSQPSDPLELVMTGAYSKPTLSALPSPLVTSEKSVTLLCQSRSPMDTFLLIKERAAHPLLHLRSEHGAQQHQAEFPMSPVTSVHGGTYRCFSSHGFSHYLLSHPSDPLELIVSGSLEDPRPSPTRSVSTAAGPEDQPLMPTGSVPHSGLRRHWE (SEQ ID NO:89).

A polypeptide comprising the amino acid sequence of:QHWRQGKHRTLAQRQADFQRPPGAAEPEPKDGGLQRRSSPAADVQGENFCAAVKDTQPEDGVEMDTRSPHDEDPQAVTYAKVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSFTLRQKATEPPPSQEGASPAEPSVYATLAIH (SEQ ID NO:90).

An embodiment of the invention is directed to a composition comprising aLIRION polypeptide sequence of SEQ ID NO:36.

A further embodiment of the invention is directed to a compositioncomprising a LIRION polypeptide fragment having biological activity ofinteracting with SHP-1, MHC I, MHC I-like molecules (such as CD1, MR1,MIC), MHCII, and complement components (such as C1, C2, C3, C4, C5, andfactor B).

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:35 encoding a LIRIONpolypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a LIRION polypeptidefragment having biological activity of interacting with SHP-1, MHC I,MHC I-like molecules (such as CD1, MR1, MIC), MHCII, and complementcomponents (such as C1, C2, C3, C4, C5, and factor B).

A further embodiment of the invention is directed to a method ofscreening test substances for modulators of LIRION expression comprisingthe steps of: i) contacting a cell with a substance to be tested; andii) comparing LIRION expression in the cell after exposure to the testsubstance to that of an untreated control cell.

A preferred embodiment relates to an antibody that specifically binds toLIRION polypeptides. Further included are LIRION binding antibodyfragments. Preferably, the LIRION-specific antibody or LIRION-bindingfragment thereof binds LIRION and not ILT3. Monoclonal, polyclonal,heterologous, and detectably labeled antibodies and antigen-bindingfragments are included in the invention. Methods of making suchantibodies and fragments thereof are common to the art and includedherein.

An additional preferred embodiment of the invention is a method ofbinding LIRION polypeptides with a LIRION-specific antibody orLIRION-binding fragment thereof. This method comprises the step ofcontacting a LIRION polypeptide with a LIRION-binding antibody orLIRION-binding fragment thereof under conditions that allow binding.This method may be applied to detection and purification of LIRION, aswell as targeting LIRION-expressing cells. These aspects are discussedin detail herein.

In one embodiment, a sequence encoding SEQ ID NO:36 bearing G to A, G toA and A to G substitutions at nucleotide positions 447, 705 and 1040 ofSEQ ID NO:35 corresponding to positions 137, 223 and 335, and resultingin the substitution of a glycine residue by a glutamic acid at position137, a glycine by an aspartic acid at position 223 and an asparagineresidue by an aspartic acid at position 335, respectively, can be usedfor DNA genotyping. Genotyping this locus could be of interest, e.g., inDNA fingerprinting for paternity studies or forensic analyses. It couldalso be used for genetic association studies, especially in pathologiesrelating to B cell autoimmune disorders (e.g., rheumatoid arthritis andulcerative colitis) and antigen presentation disorders (such as barelymphocyte syndrome).

In another embodiment, the polynucleotide sequence of the invention canbe used in pharmacogenomic applications in order to aid in the choice ofthe ideal drug (e.g. an agonist or an antagonist of LIRION), or dosageof a drug, for the treatment of a condition or disease in a patient. Forexample, in one embodiment, the invention provides a method ofgenotyping a patient to determine the identity of the nucleotidesencoding the amino acids at positions 137, 223 and 335 of LIRION, andadministering to the patient a drug or a dosage of the drug that hasbeen established to be preferentially efficacious in those with glutamicacid, aspartic acid and aspartic acid residues at positions 137, 223 and335 (e.g. because of preferential binding of the drug to the isoform ofthe protein with glutamic acid, aspartic acid and aspartic acid at thesepositions). In another embodiment, the patient is genotyped for thenucleotides encoding amino acid positions 137, 223 and 335, a drug isnot administered, e.g. because side effects are known to be associatedwith the administration of the drug to individuals with glutamic acid,aspartic acid and aspartic acid at positions 137, 223 and 335,respectively.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a LIRION polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing LIRION expression.Preferably, the polynucleotides capable of directing LIRION expressionare located in the 5′ regulatory region of the LIRION gene. Furtherpreferably, these polynucleotides are located within 500 base pairs ofthe LIRION coding region. These polynucleotides preferably comprise apromoter sequence. Techniques known in the art for introducingpolynucleotide sequences to endogenous sequences are described in U.S.Pat. No. 5,641,670 and PCT WO9629411, which disclosures are herebyincorporated by reference in their entireties.

Another embodiment relates to methods of producing LIRION polypeptides.The protein of the invention can be produced in host cells that havebeen transfected with an appropriate expression vector comprising anucleic acid sequence coding for LIRION. Introduction of such expressionvector to a host can be performed in a variety of ways, includingcalcium or lithium chloride treatment, electroporation, or lipofection.Any of a wide variety of expression systems can be used to express therecombinant proteins. Suitable expression vehicles include, but are notlimited to plasmids, viral particles or baculovirus for insect cells.The expression vehicle can be integrated into the host cell genome.Optionally, an inducible expression vector can be used to achievecontrolled expression of the gene in the host cell. The recombinantprotein can be recovered from the host cell and purified by anytechnique well known to those skilled in the art. Preferably, anantibody directed against the protein of the invention or part thereofcan be bound to a chromatographic support to form an affinitychromatography column. Alternatively, LIRION or fragments thereof can bechemically synthesized using solid-phase techniques.

In another embodiment, the present protein can be used to purify myeloidAPCs from a biological sample or from cells grown in vitro. In one sucha method a monoclonal or polyclonal antibody directed against theprotein of the invention can be used to immunopurify the cells. Methodsused to immunopurify cells are well known in the art. For example, theantibody can be fixed on a sepharose column or linked to a solidsupport. Alternatively, the antibody is fluorescently labeled and cellsare purified by FACS.

In another embodiment, the invention provides compositions and methodsfor detecting the level of expression of the mRNA encoding the proteinof the invention in a mammal, preferably a human. Quantification of mRNAlevels of LIRION may be useful for the diagnosis of conditionscorrelated with abnormal expression of the protein, including autoimmunedisorders, or to monitor regulation of LIRION during therapeuticintervention as described herein. Assays for the detection andquantification of mRNA are well known in the art. Preferred methodcomprises the steps of: isolating RNA from a biological sample,measuring LIRION mRNA level by quantitative RT-PCR, and comparing theexpression in the subject sample to that of a control sample.Polynucleotide probes or primers used for the detection of LIRION mRNAby hybridization or PCR amplification may be designed from thepolynucleotide of SEQ ID NO:35 by methods well known in the art.

In another embodiment, the invention relates to methods and compositionsfor detecting LIRION and quantifying its level of expression in abiological sample. These methods may be useful for the diagnosis ofconditions characterized by altered or abnormal LIRION expressionincluding autoimmune (e.g., rheumatoid arthritis, multiple sclerosis,and virally-induced autoimmunity), metabolic (e.g., obesity,hypercholesterolemia, insulin-related disorders), and neurological(e.g., schizophrenia) conditions. A method to quantify the expression ofLIRION protein comprises the step of: contacting a biological samplewith a LIRION-specific antibody or LIRION specific fragment thereofunder conditions that allow antibody-antigen binding. Preferably, theantibody or fragment is detectably labeled. Preferred labels includefluorescent labels (e.g., GFP, FITC, and rhodamine) and secondary labels(e.g., biotin and enzymatic substrates). A preferred method comprisescontacting an antibody which specifically binds to LIRION with abiological sample from a mammalian subject, and determining the level ofLIRION in the subject sample compared to a control level of a healthysubject, wherein abnormal level of LIRION in the subject sampleindicates that the subject has the disease or is at an elevated risk ofdeveloping the disease. The antibody used can be either monoclonal orpolyclonal and can be labeled directly or indirectly for quantificationof immune complexes by methods well known to those skilled in the art,for example by ELISA or radioimmunoassays. Preferred specific antibodiesdirected to LIRION are generated using the polypeptide fragments listedabove. Diagnostic assays to detect the protein of the invention may relyon a biological sample of a biopsy or in situ assay of cells from organor tissue sections. Preferred biological samples include whole cells andcellular extracts from organs, tissues, or blood.

An embodiment of the invention provides for a method of screening testsubstances for modulators of LIRION expression. This method comprisesthe steps of: i) contacting a cell with a test substance; ii) detectingexpression of LIRION; and iii) comparing LIRION expression in the cellafter exposure to the test substance to that of an unexposed controlcell. LIRION expression is determined by methods common to the art orincluded herein, by detecting LIRION polynucleotides or polypeptides. Anexample of this method comprises the steps of: i) culturing twoequivalent cell samples; ii) adding a test substance to one of thecultures and not the other; iii) harvesting both cultures at a specifiedtime; iv) purifying the mRNA from each sample of cells; v) detecting thelevel of LIRION mRNA in each sample. LIRION mRNA may be detected byNorthern blot, RT-PCR, or another method common to the art. Theinvention provides for design and use of specific polynucleotide probesand primers, as discussed herein. An additional example comprises thesteps of: i) having two equivalent cultures of cells; ii) adding a testsubstance to one of the cultures and not the other; iii) harvesting bothcultures; iv) purifying the protein from each sample of cells; v)detecting the level of LIRION polypeptides in each sample. LIRION may bedetected by enzyme-linked immunoabsorbent assay (ELISA), western blot,radioimmunoassay (RIA), or another method common to the art. Theinvention provides for design and use of specific antibodies andantibody fragments, as discussed herein.

A preferred embodiment of the invention provides a method of screeningfor test substances that bind LIRION polypeptides. This method comprisesthe steps of: i) contacting a test substance with a LIRION polypeptideor fragment thereof under conditions that allow binding; and ii)detecting the binding of the test substance by methods common to the art(e.g., competitive antibody-based methods such as coimmunopreciptationand Western blotting). Included in this method are test substances thatare conjugated to an antibody, antibody fragment, cell-type specificligand or a portion thereof. Substances that bind specifically to LIRIONmay be used to detect and purify the protein, as well as inhibit oractivate its activity.

A further preferred embodiment of the invention provides a method ofscreening test substances that bind to LIRION for agonists of LIRIONactivity, comprising: i) contacting a cell with the substance to betested; and ii) comparing LIRION biological activity after exposure tothe test substance to that of an unexposed control cell. Measure ofLIRION biological activity may be assessed indirectly by monitoringsignalling events following co-engagement with an activating receptor.An example of an assay to measure LIRION activation comprises the stepsof culturing monocytes in vitro, contacting the cells with the testsubstance, an anti-LIRION antibody and an anti-HLA-DR antibody, adding across-linking antibody, harvesting the cells, separating cell extractsby SDS-PAGE and detecting protein tyrosine phosphorylation using aspecific antiphosphotyrosine antibody. A decrease in tyrosine proteinphosphorylation in the test sample compared to the control sample beingindicative of an activating effect of the test substance on LIRION.Activation of LIRION can also be assessed by measuring intracellularCa++mobilization induced upon receptor co-ligation as described in Celiaet al. (1997, Journal of Experimental Medicine; Vol. 185, pp 1743-1751,which disclosure is hereby incorporated by reference in its entirety).

A further preferred embodiment of the invention provides a method ofscreening test substances that bind to LIRION for antagonists of LIRIONactivity. This method comprises the steps of: i) contacting a cell witha test substance; and ii) comparing LIRION biological activity afterexposure to the test substance to that of an unexposed control cell.Detection of LIRION biological activity may be detected by measuringprotein dephosphorylation following LIRION stimulation as mentionedabove. In this case, an increase in protein phosphorylation in the testsample compared to the control sample is indicative of an inhibitoryeffect of the test substance on LIRION activity.

Substances that increase LIRION expression or activity (agonists oractivators) may be used to decrease APC activity or treat myeloidcancers. Substances that decrease LIRION expression or activity(antagonists or inhibitors) may be used to treat or prevent infectiousdiseases.

Another embodiment of the invention is also directed to methods to treator prevent disorders associated with suppressed immune function such aspathogenic infections. This method comprises the step of introducing aLIRION antagonist to a cell. Preferred cells are those that expressLIRION, such as APCs.

A preferred embodiment of the invention includes methods of increasingLIRION expression or activity by administering a LIRION agonist orpolypeptides encoding LIRION to a cell. These methods may be applied toLIRION-associated conditions, including autoimmune (e.g.,osteoarthritis, Crohn's disease, Grave's disease, autoimmunethyroiditis, lupus erythematosus, autoimmune hemolytic anemia,thrombocytopenia, atherosclerosis, osteoporosis, asthma, myastheniagravis, rheumatoid arthritis, multiple sclerosis, and virally-inducedautoimmunity), metabolic (e.g., obesity, hypercholesterolemia,insulin-related disorders), and neurological (e.g., schizophrenia)conditions as well as myeloid cancers. One such method comprises thestep of contacting a cell with a LIRION agonist. Preferred agonistsinclude those that affect LIRION expression, such as heterologouspromoter sequences. An additional method comprises the step ofintroducing a polynucleotide encoding a LIRION polypeptide to a cell.One example of this method includes: i) removing a sample of APC cellsof an individual; ii) introducing ex vivo a polynucleotide sequencecomplementary to LIRION polynucleotide sequences to those cells, andiii) reinjecting the recombinant cells into the individual. APCs includemonocytes, macrophages and dendritic cells. Expression vectors derivedfrom retroviruses, adenovirus, herpes, or vaccinia viruses may be usedfor the delivery of the polynucleotide sequences complementary to thoseof the invention to the targeted cell population. Many methods forintroducing vectors into cells or tissues are available and equallysuitable for use either in vivo or ex vivo. Delivery by transfection andliposome injections may be achieved using methods which are well knownin the art. Pharmaceutical compositions comprising LIRION agonists,antagonists, or oligonucleotides can be administered alone or incombination with at least one other agent, such as stabilizing compound,which may be administered in any sterile biocompatible carrier. They maybe administered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventiculary, transdermal, subcutaneous, intranasal,enteral or rectal means. The compositions may be administered to thesubject alone or in combination with other agents, or drugs.

It is yet a further object of the invention to provide compositions andmethods to deliver into a cell a molecule of interest comprisingcontacting cells expressing LIRION with a molecule joined to a LIRIONantibody. Preferably, these methods are to be applied in vivo. Apreferred method comprises joining a molecule of interest with acompound that specifically binds the extracellular portion of LIRION;and contacting the chimeric molecule with a cell that express underconditions thates trigger LIRION internalization. The targeted cellpopulations may be naturally expressing the protein of the invention attheir cell surface or may be transfected by LIRION encoding sequencesprior to the addition of the chimeric antibody. Antibodies to be used insuch methods are directed against the extracellular domain of the matureprotein, preferably comprising the epitope VTSEKSVT or a LIRION-specificfragment thereof. Molecules to be delivered into the cells can befluorescent or radioactive dyes or therapeutically active molecules,including but not limited to chemical drugs, radioactive compounds,peptides or nucleic acids. Methods to link an antibody moiety to asecond molecule are well known by those skilled in the art.Alternatively, chimeric proteins with LIRION antibodies may also beobtained by synthesis in vivo. Using these methods would be ofparticular interest in gene therapy, radioimmunoscintigraphy (RIS) andradioimmunotherapy (RIT) of specific LIRION-expressing cell types suchas APCs.

Protein of SEQ ID NO:38 (Internal Designation Clone 588394160-105-4-0-A1-F)

The cDNA of Clone 588394 160-105-4-0-A11-F (SEQ ID NO:37) encodes the383 amino acid SLAMP protein of SEQ ID NO:38 comprising the amino acidsequence: MRTYWLHSVWVLGFFLSLFSLQGLPVRSVDFNRGTDNITVRQGDTAILRCVVEDKNSKVAWLNRSGIIFAGHDKWSLDPRVELEKRHSLEYSLRIQKVDVYDEGSYTCSVQTQHEPKTSQVYLIVQVPPKISNISSDVTVNEGSNVTLVCMANGRPEPVITWRHLTPTGREFEGEEEYLEILGITREQSGKYECKAANEVSSADVKQVKVTVNYPPTITESKSNEATTGRQASLKCEASAVPAPDFEWYRDDTRINSANGLEIKSTEGQSSLTVTNVTEEHYGNYTCVAANKLGVTNASLVLFKRVLPTIPHPIQEIGTTVHFKQKGIFLSESQRGETTKITLNCGNLFLRNLHPTSDQEPQRLWTLCCLLPRKGQHRIYGQC. Accordingly, it will be appreciated that allcharacteristics and uses of polypeptides of SEQ ID NO:38 describedthroughout the present application also pertain to the polypeptidesencoded by the human cDNA included in Clone 588394 160-105-4-0-All-F. Inaddition, it will be appreciated that all characteristics and uses ofthe polynucleotides of SEQ ID NO:37 described throughout the presentapplication also pertain to the nucleic acids comprising the human cDNAin 588394 160-105-4-0-A11-F. Also preferred are fragments having abiological activity as described therein and the polynucleotidesencoding the fragments.

SLAMP, the protein of SEQ ID NO:38, represents a novel splice variant ofthe limbic system-associated membrane protein (LAMP; Swissprot entryQ13449). SLAMP has a 22 amino acid signal sequence(MRTYWLHSVWVLGFFLSLFSLQ) (SEQ ID NO:91), three C2-typeimmunoglobulin-like-domains, and a unique 80 amino acidcarboxy-terminus. The protein belongs to the IgLON family, a group ofneuronal glycoproteins which have been isolated from chicken, rat andhuman. These include the LAMP protein, Neurotrimin/CEPU-1, OBCAM andkilon/Neurotractin polypeptides. Most of the known members of thisfamily are cell-surface adhesion molecules with a glycosylphosphatidylinositol anchor at their C-terminus which tethers them tothe neuronal plasma membrane. SLAMP, however, lacks the GPI anchor andis thus secreted by neurons in cortical and subcortical regions of thelimbic system. SLAMP interacts with itself and other IgLON proteins andis involved in cell-cell recognition, contact-dependent regulation ofneurite out-growth and axon guidance of specific subset of neuronsduring brain development. SLAMP interacts with LAMP, Neurotrimin andOBCAM, promotes neurite outgrowth of limbic neurons and inhibits neuriteoutgrowth of non-limbic neurons both in vivo and in vitro.

Preferred SLAMP polypeptides for uses in the methods described belowinclude the polypeptides comprising the amino sequence of: (SEQ IDNO:92) GLPVRSVDFNRGTDNITVRQGDTAILRCVVEDKNSKVAWLNRSGIIFAGHDKWSLDPRVELEKRHSLEYSLRIQKVDVYDEGSYTCSVQTQHEPKTSQVYLIVQVPPKISNISSDVTVNEGSNVTLVCMANGRPEPVITWRHLTPTGREFEGEEEYLEILGITREQSGKYECKAANEVSSADVKQVKVTVNYPPTITESKSNEATTGRQASLKCEASAVPAPDFEWYRDDTRINSANGLEIKSTEGQSSLTVTNVTEEHYGNYTCVAANKLGVTNASLVLFKRVLPTIPHPIQEIGTTVHFKQKGIFLSESQRGETTKITLNCGNLFLRNLHPTSDQEPQRLWTLCCLLP RKGQHRIYGQC;

A polypeptide comprising the amino acid sequence of: (SEQ ID NO:93)GLPVRSVDFNRGTDNITVRQGDTAILRCVVEDKNSKVAWLNRSGIIFAGHDKWSLDPRVELEKRHSLEYSLRIQKVDVYDEGSYTCSVQTQHEPKTSQVYLIVQVPPKISNISSDVTVNEGSNVTLVCMANGRPEPVITWRHLTPTGREFEGEEEYLEILGITREQSGKYECKAANEVSSADVKQVKVTVNYPPTITESKSNEATTGRQASLKCEASAVPAPDFEWYRDDTRINSANGLEIKSTEGQSSLTVTNVTEEHYGNYTCVAANKLGVTNASLVLF;

A polypeptide comprising the amino acid sequence of: (SEQ ID NO:94)KRVLPTIPHPIQEIGTTVHFKQKGIFLSESQRGETTKITLNCGNLFLRNLHPTSDQEPQRLWTLCCLLPRKGQHRIYGQC.

An embodiment of the invention is directed to a composition comprising aSLAMP polypeptide sequence of SEQ ID NO:38.

A further embodiment of the invention is directed to a compositioncomprising a SLAMP polypeptide fragment having biological activity ofbinding to any one of the group consisting of LAMP, Neurotrimin, CEPU-1,CEPU-1-Se, OBCAM, kilon, Neurotractin, and GP55-A.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:37 encoding a SLAMPpolypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a SLAMP polypeptidefragment having biological activity of binding to any one of the groupconsisting of LAMP, Neurotrimin, CEPU-1, CEPU-1-Se, OBCAM, kilon,Neurotractin, and GP55-A.

A further embodiment of the invention is directed to a method ofscreening test substances for modulators of SLAMP expression comprisingthe steps of: i) contacting a cell with a substance to be tested; andii) comparing SLAMP expression in the cell after exposure to the testsubstance to that of an untreated control cell.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a SLAMP polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing SLAMP expression.Preferably, the polynucleotides capable of directing SLAMP expressionare located in the 5′ regulatory region of the SLAMP gene. Furtherpreferably, these polynucleotides are located within 500 base pairs ofthe SLAMP coding region. These polynucleotides preferably comprise apromoter sequence.

Techniques known in the art for introducing polynucleotide sequences toendogenous sequences are described in U.S. Pat. No. 5,641,670 and PCTWO9629411, which disclosures are hereby incorporated by reference intheir entireties.

One embodiment of the present invention relates to methods of producingSLAMP polypeptides. The protein of the invention can be produced in hostcells that have been transfected with an appropriate expression vectorcomprising a nucleic acid sequence coding for the protein of theinvention. Introduction into a host cell of such expression vector forSLAMP can be performed in a variety of ways, including but not limitedto calcium or lithium chloride treatment, electroporation, orlipofection. Any of a wide variety of expression systems can be used toprovide the recombinant proteins. Suitable expression vehicles include,but are not limited to plasmids, viral particles or baculovirus forinsect cells. The expression vehicle can be integrated into the hostcell genome. Optionally, an inducible expression vector can be used toachieve tight controlled expression of the gene in the host cell. Therecombinant protein can be recovered from the host cell or the cellculture medium and purified by any technique well known to those skilledin the art. Preferably, a SLAMP-specific antibody SLAMP-specificfragment thereof can be bound to a chromatographic support to form anaffinity chromatography column. Alternatively, SLAMP or fragmentsthereof can be chemically synthesized using solid-phase techniques.

In a further embodiment, the invention provides compositions and methodsusing SLAMP or fragments thereof to label neurons in order to visualizeany change in number, topology or morphology of these cells. Preferredneurons are those expressing LAMP, Neurotrimin, CEPU-1, CEPU-1-Se,OBCAM, kilon, Neurotractin, and GP55-A. These changes may be associatedwith a central nervous system disorder. SLAMP may be rendered easilydetectable by labeling or conjugating with a detectable molecule orantibody.

In another embodiment, the present protein can be used to purifyneuronal cells from a biological sample such as neurons grown in vitro.This method comprises the steps of: i) contacting a cell sample withSLAMP or a biologically active fragment thereof, ii) removingnon-binding contaminants; and iii) eluting the remaining cells.Preferred cell samples are samples comprising neuronal cells. Furtherpreferred are cells expressing LAMP, Neurotrimin, CEPU-1, CEPU-1-Se,OBCAM, kilon, Neurotractin, and GP55-A. Preferably, SLAMP is fixed on asepharose column or covalently or noncovalently linked to a solidsupport. Alternatively, the neuronal cells can be preincubated withSLAMP and then a monoclonal or polyclonal antibody directed againstSLAMP can be used to immunopurify the cells, for example on a column.Alternatively, the SLAMP-specific antibody is fluorescently labeled andcells are purified by FACS.

In another embodiment, the invention provides compositions and methodsfor detecting the level of expression of the mRNA encoding the proteinof the invention in a mammal, preferably a human. This method comprisesthe steps of: contacting a SLAMP mRNA or cDNA molecule with a detectablecompound under conditions that allow binding. Preferably, the detectablecompound is a nucleotide sequence complimentary to SLAMP mRNA.Preferably, SLAMP mRNA is purified from a cell sample. SLAMP cDNA ispreferably generated from such an RNA sample. Quantification of mRNAlevels of SLAMP may be useful for the diagnosis of diseases orconditions correlated with abnormal expression of the protein, or tomonitor regulation of SLAMP during therapeutic intervention as describedherein. Assays for the detection and quantification of mRNA are wellknown in the art. An example of this method comprises the steps of:isolating RNA from a brain sample of a subject, measuring SLAMP mRNAlevel by quantitative RT-PCR, and comparing the expression in thesubject sample to that of a control sample. Polynucleotide probes orprimers used for the detection of SLAMP mRNA by hybridization or PCRamplification may be designed from the polynucleotide of SEQ ID NO:37 bymethods well known in the art.

In another embodiment, the invention relates to methods and compositionsfor detecting SLAMP and quantifying its level of expression in abiological sample. These methods may be useful for the diagnosis ofconditions or diseases characterized by altered or abnormal expressionof the protein of the invention, or in assays to monitor subjects beingtreated with SLAMP, agonists or antagonists. A preferred methodcomprises contacting an antibody which specifically binds to SLAMP witha brain sample from a mammalian subject, preferably human, anddetermining the level of SLAMP in the subject sample compared to acontrol level representative of a healthy subject, wherein an altered orabnormal level of SLAMP in the subject sample relative to the controllevel indicates that the subject has the disease or is at an elevatedrisk of developing the disease. The antibody used can be eithermonoclonal or polyclonal and can be labeled directly or indirectly forquantification of immune complexes by methods well known to thoseskilled in the art, for example by ELISA or radioimmunoassays. Preferredspecific antibodies directed to SLAMP are generated using thepolypeptide fragments listed above. Diagnostic assays to detect theprotein of the invention may comprise a biopsy, in situ assay of cells.In addition, assays may be conducted upon cellular extracts.

An embodiment of the invention provides for a method of screening testsubstances for modulators of SLAMP expression. This method comprises thesteps of: i) contacting a cell with a test substance; and ii) comparingSLAMP expression in the cell after exposure to the test substance tothat of an unexposed control cell. SLAMP expression is determined bymethods common to the art or included herein, by detecting SLAMPpolynucleotides or polypeptides. An example of this method comprises thesteps of: i) culturing two equivalent cell samples; ii) adding a testsubstance to one of the cultures and not the other; iii) harvesting bothcultures at a specified time; iv) purifying the mRNA from each sample ofcells; and v) detecting the level of SLAMP mRNA in each sample.Detection may be accomplished by Northern blot, RT-PCR, or anothermethod common to the art. The invention provides for design and use ofspecific polynucleotide probes and primers, as discussed herein. Anadditional example comprises the steps of: i) having two equivalentcultures of cells; ii) adding a test substance to one of the culturesand not the other; iii) harvesting both cultures; iv) purifying theprotein from each sample of cells; and v) detecting the level of SLAMPpolypeptides in each sample. Detection may be accomplished byenzyme-linked immunoabsorbent assay (ELISA), western blot,radioimmunoassay (RIA), or another method common to the art. Theinvention provides for design and use of specific antibodies andantibody fragments, as discussed herein. Substances that increase SLAMPexpression or activity (agonists or activators) may be used to treat orprevent neurodegenerative diseases affecting the limbic system orabnormal cell proliferation in the non-limbic region. Substances thatdecrease SLAMP expression or activity (antagonists or inhibitors) may beused to treat or prevent conditions associated with abnormal cellproliferation in the limbic region or neurodegenerative diseasesaffecting primarily the non limbic system. Methods utilizing SLAMPagonists and antagonists are included herein.

A preferred embodiment of the invention provides a method of screeningfor test substances that bind SLAMP polypeptides. This method comprisesthe steps of: i) contacting a test substance with a SLAMP polypeptide orfragment thereof under conditions that allow binding; and ii) detectingthe binding of the test substance by methods common to the art (e.g.,competitive antibody-based methods such as coimmunopreciptation andWestern blotting). Included in this method are test substances that areconjugated to an antibody, antibody fragment, cell-type specific ligandor a portion thereof. This method may be used for purification anddetection of SLAMP polypeptides, as well as for the screening methodsdiscussed below.

A further preferred embodiment of the invention provides a method ofscreening test substances that bind to SLAMP for agonists of SLAMPactivity. This method comprises the steps of: i) contacting a cell withSLAMP and the substance to be tested; and ii) comparing SLAMP biologicalactivity after exposure to the test substance to that of a controlexposed only to SLAMP. Measure of SLAMP biological activity may beassessed by monitoring neurite outgrowth. An example of an in vitroassay to measure SLAMP activation comprises the steps of culturinghippocampal neurons in vitro, contacting the cells with SLAMP and thetest substance, and measuring neurite number/cell or neurite extensionas described by Marg et al. (1999; Journal of Cell Biology, vol. 145, pp865-876, which disclosure is hereby incorporated by reference in itsentirety), an increase in the test sample compared to the control samplebeing indicative of an activating effect of the test substance on SLAMP.

A further preferred embodiment of the invention provides a method ofscreening test substances that bind to SLAMP for antagonists of SLAMPactivity. This method comprises the steps of: i) contacting a cell withSLAMP and a test substance; and ii) comparing SLAMP biological activityafter exposure to the test substance to that of an unexposed control.SLAMP activity may be measured as described above, a decrease in SLAMPactivity in the test sample compared to the control sample beingindicative of an inhibitory effect of the test substance on SLAMP.

Another embodiment of the invention is also directed to methods to treator prevent a disease state of a mammal, preferably a human, associatedwith excessive neural growth in the limbic region comprisingadministering to said mammal antagonistic antibodies, ribozymes orantisense vectors or oligonucleotides. Preferably, the disease state tobe treated by such methods is brain cancer. A preferred method comprisesinjecting into an individual antagonistic antibodies directed againstSLAMP. Polynucleotide or oligonucleotide sequences complementary toSLAMP polynucleotide sequences may also be used to decrease or inhibitSLAMP expression by limbic neurons. Expression vectors derived fromretroviruses, adenovirus, herpes, or vaccinia viruses may be used forthe delivery of the polynucleotide sequences complementary to those ofthe invention to the targeted cell population. Many methods forintroducing vectors into cells or tissues are available and equallysuitable for use either in vivo or ex vivo. Delivery by transfection andliposome injections may be achieved using methods which are well knownin the art. Alternatively, an antagonist of SLAMP expression or activityisolated as described above can be used, such method comprising the stepof administering to a subject an antagonist of SLAMP expression oractivity. Preferably, the antagonist is delivered to the limbic neuronsof the subject, for example, by conjugating the antagonist to a cellspecific targeting moiety (e.g., an antibody fragment). Pharmaceuticalcompositions comprising SLAMP agonists, antisense vectors oroligonucleotides can be administered alone or in combination with atleast one other agent, such as stabilizing compound, which may beadministered in any sterile biocompatible carrier. They may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventiculary, transdermal, subcutaneous, intranasal,enteral or rectal means. The compositions may be administered to thesubject alone or in combination with other agents, or drugs.

It is another object of the invention to provide methods for treating orpreventing neuropathologies in a mammal, preferably human, involving thelimbic system. This can be achieved by increasing SLAMP expression oractivity in vivo using the protein of the invention or fragments thereofor polynucleotides encoding the present protein. A preferred methodcomprises introducing in vivo into a subject an effective amount ofSLAMP polypeptides with a pharmaceutically acceptable carrier. Increasedexpression of the protein of the invention may also be achieved byintroducing into the cells of a subject the sequences encoding theprotein of the invention. A preferred method ex-vivo comprising: i)introducing to neural stem cells a polynucleotide sequence encodingSLAMP, and ii) injecting the recombinant cells into an individual.Alternatively, an agonist of SLAMP expression or activity isolated asdescribed above can be used, such method comprising the step ofadministering to a subject an agonist of SLAMP expression or activity.SLAMP agonists in a physiologically acceptable solution may be deliveredby methods common to the art. Preferably, the SLAMP agonist is deliveredin vivo to the limbic neurons of the subject. These method are usefulfor prevention and treatment of neurodegenerative diseases andneuropathies including, but not limited to, Alzeimer's disease,Parkinson's disease, Huntington's disease, cerebral ischaemia, limbicencephalitis, cerebellar ataxia, schizophrenia, and Tourette syndrome.

Protein of SEQ ID NO:40 (Internal Designation Clone 495718160-26-2-0-E12-F)

The cDNA of Clone 495718_(—)160-26-2-0-E12-F (SEQ ID NO: 39) encodesSAP-MU-10 of SEQ ID NO:40, comprising the amino acid sequence:MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTASDCGAVKHCLQTVWNKPTVKSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESLQKHLAELNHQKQLESNKIPELDMTEVVAPFMANIPLLLYPQDGPRSKPQPKDNGDVCQDCIQMVTDIQTAVRTNSTFVQALVEHVKEECDRLGPGMADICKNYISQYSEIAIQMMMHMQDQQPKEICALVGFCDEVKEMPMQTLVPAKVASKNVIPALELVEPIKKHEVPAKSDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGTRLPALTVHVTQPKDGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVWILPSCA. Accordingly, itwill be appreciated that all characteristics and uses of thepolypeptides of SEQ ID NO:40 described throughout the presentapplication also pertain to the polypeptides encoded by the human cDNAincluded in Clone 495718_(—)160-26-2-0-E12-F. In addition, it will beappreciated that all characteristics and uses of the polynucleotides ofSEQ ID NOs:39 described throughout the present application also pertainto the nucleic acids comprising the human cDNA in Clone495718_(—)160-26-2-0-E12-F. A preferred embodiment of the invention isdirected toward the compositions of SEQ ID NO:40, SEQ ID NO:39, andClone 495718_(—)160-26-2-0-E12-F. Preferred SAP-MU-10 polypeptidefragment for uses in the methods described below include the Sap-D10polypeptide comprising the amino sequence of:DGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVWILPSCA (SEQ ID NO:95). Also preferred are polypeptide fragmentscomprising the seven C-terminal amino acids, having a biologicalactivity as described herein and the polynucleotides encoding thefragments.

The protein of the invention, SAP-MU-10, is a novel isoform of Saposin(GenBank accession number P07602). Three different isoforms of Saposinhave been described. The 472 amino terminal amino acids of SAP-MU-10 areidentical to the SAP-MU-9 isoform and the 7 carboxyl-terminalamino-acids are unique to SAP-MU-10.

SAP-MU-10 is a precursor for 4 different proteins, and proteolyticcleavage of SAP-MU-10 gives rise to the Sap-A10, Sap-B10, Sap-C10 andSap-D10 proteins. The unprocessed SAP-MU-10 precursor binds tosphingolipids and promotes ganglioside transport from liposomes tomembrane. The processed proteins are non-enzymatic activators of thehydrolysis of sphingolipids. Sphingolipids are important components ofthe myelin sheath, a structure that protects and insulates nerve fibers.SAP-MU-10 and Sap-D10 play a major role both in myelination of neuronsand in storage of sphingolipids. SAP-MU-10 is expressed and processed invarious tissues, being expressed at especially high level in the nervoussystem. Furthermore, the unprocessed SAP-MU-10 precursor is secreted inthe cerebrospinal plasma. The SAP-MU-10 protein binds to sphingolipidsand gangliosides, and displays neurotrophic properties. The Sap-D10protein binds to sphingomyelin and ceramide, and displays asphingomyelin phosphodiesterase activator activity.

As used herein, SAP-MU-10 refers to the polypeptide sequence of SEQ IDNO:40. As used herein, a SAP-MU-10 polypeptide refers to a fragment ofpolypeptide sequence of SEQ ID NO:40 having either sphingolipid organglioside binding activity, or neurotrophic properties. As usedherein, a Sap-D10 polypeptide refers to a polypeptide comprising theSap-D10 polypeptide described above and having sphingomyelinasephosphodiesterase activator activity.

An embodiment of the invention is directed to a composition comprising aSAP-MU-10 polypeptide sequence of SEQ ID NO:40.

A further embodiment of the invention is directed to a compositioncomprising a SAP-MU-10 polypeptide fragment having biological activity.

A further embodiment of the invention is directed to a compositioncomprising a Sap-D10 polypeptide fragment having biological activity.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:39 encoding aSAP-MU-10 polypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:39 encoding a Sap-D10polypeptide.

A further embodiment of the invention is directed to a compositioncomprising an antibody that specifically recognizes a SAP-MU-10polypeptide. Preferably, the antibody recognizes an epitope comprisingone or more of the 7 C-terminal amino acids of SAP-MU-10 and Sap-D10,wherein one or more of the seven C-terminal amino acids is required forantibody binding. Preferably, the antibody binds specifically toSAP-MU-10 and to Sap-D10 but not to other saposin isoforms.Alternatively, the antibody binds specifically to SAP-MU-10 but not toSap-D10 and other saposin isoforms. Alternatively, the antibody bindsspecifically to Sap-D10 but not to SAP-MU-10 and other saposin isoforms.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a SAP-MU-10 polypeptide or a biologicallyactive fragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing SAP-MU-10expression. Preferably, the polynucleotides capable of directingSAP-MU-10 expression are located in the 5′ regulatory region of theSAP-MU-10 gene. Further preferably, these polynucleotides are locatedwithin 500 base pairs of the SAP-MU-10 coding region. Thesepolynucleotides preferably comprise a promoter sequence. Techniquesknown in the art for introducing polynucleotide sequences to endogenoussequences are described in U.S. Pat. No. 5,641,670 and PCT WO9629411,which disclosures are hereby incorporated by reference in theirentireties. SAP-MU-10 protein produced by said host cell may be used forin vitro detection and purification methods as well as diagnosis and invivo applications.

Another embodiment relates to a method of producing SAP-MU-10polypeptides comprising the steps of: i) transfecting a mammalian hostcell with a recombinant expression vector comprising a polynucleotide ofthe present invention, and ii) purifying the produced protein. Thepurification of the protein can be done following any techniquewell-known to those skilled in the art. Preferably, an antibody directedagainst SAP-MU-10 or part thereof may be bound to a chromatographicsupport to form an affinity chromatography column.

An embodiment of the invention is a method comprising the step ofbinding a SAP-MU-10 polypeptide with a sphingolipid or a gangliosidemolecule under conditions that allow binding of a SAP-MU-10 polypeptideto said molecule. In a preferred embodiment, a SAP-MU-10 polypeptide isused to purify sphingolipid or ganglioside molecules. In such method, aSAP-MU-10 polypeptide is preferably covalently or non-covalentlyattached to a solid matrix and allowed to bind sphingolipids andgangliosides using techniques well-known in the art. This methodcomprises the steps of: i) contacting SPA-MU-10 with a sphingolipid organglioside molecule in solution; ii) washing the solid matrix to removenon-binding contaminants; and iii) eluting the sphingolipid organglioside using more stringent conditions. Purifying sphingolipids andgangliosides can for example be useful for confirming deficiency oflysosomal enzyme activity affecting ganglioside breakdown, or formonitoring ganglioside catabolism in cultured fibroblasts loaded withganglioside.

Additional aspects of this embodiment include methods of using SAP-MU-10polypeptides to detect and quantify sphingolipids and gangliosides usingtechniques common in the art. Such methods comprise the steps of: i)obtaining a biological sample suspected of containing sphingolipids organgliosides; ii) contacting such sample with a SAP-MU-10 polypeptideunder conditions allowing binding of SAP-MU-10; and iii) detecting thepresence or absence of sphingolipids and gangliosides by detectingSAP-MU-10. Preferably, the SAP-MU-10 polypeptide is covalently attachedto a detectable compound (e.g., enzymatic substrates, or fluorescent,luminescent, and radioactive compounds). Alternatively, a detectableSAP-MU-10-specific antibody may be used to detect SAP-MU-10. Thisembodiment is useful, for example, as a diagnostic tool for quantifyingthe amount of sphingolipids in cerebrospinal plasma. Such diagnostictool may be useful to diagnose sphingolipidosis and various lysosomalstorage disorders (LSDs) such as, e.g., cystinosis, Gaucher's disease,multiple sulfatase deficiency, Niemann-Pick disease, Pompe's disease andWolman's disease.

In another embodiment, the invention is directed to a method ofdetecting SAP-MU-10 polypeptides in a biological sample, said methodcomprising the steps of: i) contacting a biological sample with anantibody or antigen binding antibody fragment that specifically bindsSAP-MU-10 polypeptide; and ii) detecting the antigen-antibody complexformed. The antibody or antigen binding antibody fragment may bemonoclonal or polyclonal. In addition, the antibody or antigen bindingantibody fragment may be primarily or secondarily labeled by adetectable compound (e.g., radioactive, fluorescent, luminescent, orenzymatic) common in the art.

In some embodiments, the invention also concerns a diagnostic kit fordetecting in vitro the presence of a SAP-MU-10 polypeptide. Such kitcomprises: i) a polyclonal or monoclonal antibody or fragment thereofthat specifically binds a SAP-MU-10 polypeptide; and optionally, ii) areagent allowing the detection of the antigen-antibody complexes formed.Preferably, the antibody or antibody fragment is detectably labeled.Such labels include fluorescent, luminescent, and radioactive compounds,as well as enzymatic substrates. The optional reagent may provide adetectable signal and either bind to the antibody or react with thelabel on such antibody. Preferably, the kit containing antibodies forSAP-MU-10 is used to diagnose LSDs such as those listed herein.Alternatively, the kit of the present invention is used for monitoringthe condition of a patient receiving treatment for a LSD. To detect suchdisorders, an appropriate biological sample (blood or cerebrospinalplasma for example) can be tested to determine the level of SAP-MU-10being produced. A kit for detecting LSDs comprising an anti-SAP-MU-10antibody may also comprise other antibodies such as those described inPCT application WO00/55632, which disclosure is incorporated byreference in its entirety. Alternatively, an anti-Sap-D10 antibody maybe used in such method.

An embodiment of the invention provides for a method of screening testsubstances for modulators of SAP-MU-0 expression. This method comprisesthe steps of: i) contacting a cell with a test substance; and ii)comparing SAP-MU-10 expression in the cell after exposure to the testsubstance to that of an unexposed control cell. SAP-MU-10 expression isdetermined by methods common to the art or included herein, by detectingSAP-MU-10 polynucleotides or polypeptides. An example of this methodcomprises the steps of: i) culturing two equivalent cell samples; ii)adding a test substance to one of the cultures and not the other; iii)harvesting both cultures at a specified time; iv) purifying the mRNAfrom each sample of cells; v) comparing the level of SAP-MU-10 mRNA ineach sample by Northern blot, RTPCR, or another method common to theart. The invention provides for design and use of specificpolynucleotide probes and primers, as discussed herein. An additionalexample comprises the steps of: i) having two equivalent cultures ofcells; ii) adding a test substance to one of the cultures and not theother; iii) harvesting both cultures; iv) purifying the protein fromeach sample of cells; v) comparing the level of SAP-MU-10 polypeptidesin each sample by Western blot, immunohistochemistry, or another methodcommon to the art. The invention provides for design and use of specificantibodies and antibody fragments, as discussed herein.

In another embodiment, a SAP-MU-10 polypeptide or a fragment thereof,may be used to screen for compounds that activate or inhibit SAP-MU-10activity. This method comprises the steps of: i) contacting a SAP-MU-10polypeptide or fragment thereof with a test substance, and ii)monitoring SAP-MU-10 activity. SAP-MU-10 activity may be monitored bybinding assays with gangliosides as described by Hiraiwa et al. (ProcNatl Acad Sci USA. 89:11254-8 (1992)), which disclosure is incorporatedby reference in its entirety.

In another embodiment, a Sap-D10 polypeptide may be used to screen forcompounds that activate or inhibit Sap-D10 activity. This methodcomprises the steps of: i) contacting a Sap-D10 polypeptide or fragmentthereof with a test substance, and ii) monitoring Sap-D10 activity.Sap-D10 activity may for example be determined by monitoringsphingomyelinase activity as described by Morimoto et al. (BiochemBiophys Res Commun 156:403-10 (1988)), which disclosure is incorporatedby reference in its entirety.

Test substances that decrease SAP-MU-10 expression or activity aredefined as inhibitors or antagonists of SAP-MU-10. Test substances thatdecrease Sap-D10 activity are defined as inhibitors or antagonists ofSap-D10. Test substances that increase SAP-MU-10 expression or activityor that increase Sap-D10 activity are defined as activators or agonists.Agents which modulate the expression or activity of SAP-MU-10 or Sap-D10activity of the subject invention include, but are not limited to,antisense oligonucleotides, ribozymes, and antibodies. These agents maybe made and used according to methods well known in the art.

One embodiment of the present invention relates to a method forincreasing myelination, stimulating neural cell outgrowth, or slowingdown a process of demyelination or neural degeneration. The methodcomprises the step of: contacting a cell with a composition comprising aSAP-MU-10 polypeptide or an agonist thereof. Preferred cells are neuronsand oligodendrocytes. The cells can be treated in vitro or in vivo bydirectly administering the composition to the cells. In a preferredembodiment, a composition comprising a SAP-MU-10 polypeptide or anagonist thereof is introduced to an individual suffering from ademyelinating disorder (e.g., multiple sclerosis, virus-inducedinflammatory demyelination, leukoencephalopathies and leukodystrophiessuch as Krabbe's disease, metachromatic leukodistrophy, ALD, Canavandisease and Alexander disease). Preferably, the amount of agonist orpolypeptide used is effective to increase myelination in an individual.In another preferred embodiment, a composition comprising a SAP-MU-10polypeptide or an agonist thereof is introduced to an individualsuffering from a neurodegenerative disorder (e.g., retinal neuropathy,Alzheimer's disease, Parkinson's disease, stroke, post-polio syndromeand amyotrophic lateral sclerosis). Preferably, the amount of agonist orpolypeptide used is effective to stimulate neural cell outgrowth.

In another embodiment, a Sap-D10 polypeptide or a Sap-D10 agonist isused to activate sphingomyelinase diphosphoesterase in vitro or in vivo.A preferred embodiment is directed to a method comprising the step of:contacting a sphingomyelinase diphosphoesterase with a Sap-D10polypeptide or a Sap-D10 agonist. Another preferred embodiment isdirected to a method comprising the step of: contacting a cell with acomposition comprising a Sap-D10 polypeptide or an agonist thereof.Still another preferred embodiment relates to a method comprising thestep of: administering a composition comprising a Sap-D10 polypeptide ora Sap-D10 agonist in a physiologically acceptable composition to anindividual. Preferably, such a method is directed to treat an individualsuffering from Tay-Sachs disease associated with Saposin D deficiency.Preferably, the amount of Sap-D10 agonist or polypeptide used iseffective to increase sphingomyelinase phosphodiesterase activity in theindividual suffering from Tay-Sachs disease.

The composition of the invention may comprise a pharmaceuticallyacceptable carrier such as those described herein and may beadministered to an individual by any technique known in the art.Preferably, the composition is introduced by injection (e.g., directintracranial injection, injection to the cerebrospinal fluid, localinjection to peripheral neural tissue or systemic injection). Even morepreferably, the composition is introduced in a patient by one of themethods described in U.S. Pat. No. 5,714,459, and U.S. Pat. No.4,902,505 which disclosure is incorporated by reference in its entirety.

Another embodiment is directed to a method of using SAP-MU-10polynucleotides comprising the steps of: i) constructing a recombinantmolecule comprising a nucleic acid sequence encoding a SAP-MU-10polypeptide that allows expression of SAP-MU-10 or fragment thereofunder suitable physiological conditions, and ii) introducing thisrecombinant molecules into a cell, a mammal or a human. Preferably, therecombinant molecule is introduced into a neural cell.

Recombinant molecules comprising a nucleic acid sequence encoding aSAP-MU-10 polypeptide may be directly introduced into cells or tissuesin vitro using delivery vehicles such as retroviral vectors, adenoviralvectors and DNA virus vectors. They may also be introduced into cells invivo using physiological techniques such as microinjection andelectroporation or chemical methods such as coprecipitation andincorporation of DNA into liposomes. Recombinant molecules may also bedelivered in the form of an aerosol or by lavage. The SAP-MU-10polynucleotides may also be applied intracellularly such as by directinjection into cells. Methods for carrying out SAP-MU-10 transfer into acell and grafting in the brain are described in U.S. Pat. No. 5,762,926,which disclosure is hereby incorporated by reference in its entirety.Such a method can be used to treat, e.g., multiple sclerosis,leukoencephalopathies and leukodystrophies.

Another embodiment of the invention relates to transgenic animals andmethods of using a SAP-MU-10 polynucleotide sequence or part thereof toestablish transgenic animals (D. melanogaster, M. Musculus), by anymethod familiar to those skilled in the art. By modulating in vivo theexpression of the transgene with drugs or modifier genes (activator orsuppressor genes), animal models can be developed that mimic humandisorders such as LSDs. These animal models would thus allow theidentification of potential therapeutic agents for treatment of thedisorders. In addition, recombinant cell lines derived from thesetransgenic animals may be used for similar approaches ex vivo.

Protein of SEQ ID NO:42 (Internal Designation Clone 612386187-9-4-0-B2-F)

The cDNA of Clone 612386_(—)187-9-4-0-B2-F (SEQ ID NO: 41) encodescytogram of SEQ ID NO:42, comprising the amino acid sequence:MELCRSLALLGGSLGLMFCLIALSTDFWFEAVGPTHSAHSGLWPTGHGDIISGHGPLVSTTAAFAAGKDSGLDWGIASQRIPAEELSHLSCPCPQPSPWWWPWRCTPASGGTSLHTPRSRPSSPGPSTWAGSQLSSCSVQVP. Accordingly, it will be appreciated that allcharacteristics and uses of the polypeptides of SEQ ID NO:42 describedthroughout the present application also pertain to the polypeptidesencoded by the human cDNA included in Clone 612386_(—)187-9-4-0-B2-F. Inaddition, it will be appreciated that all characteristics and uses ofthe polynucleotides of SEQ ID NOs:41 described throughout the presentapplication also pertain to the nucleic acids comprising the human cDNAin Clone 612386_(—)187-9-4-0-B2-F. A preferred embodiment of theinvention is directed toward the compositions of SEQ ID NO:42, SEQ IDNO:41, and Clone 612386_(—)187-9-4-0-B2-F. Preferred cytogrampolypeptides for uses in the methods described below include thepolypeptides comprising the amino sequence of:AHSGLWPTGHGDIISGHGPLVSTTAAFAAGKDSGLDWGIASQRIPAEELSHLSCPCPQPSPWWWPWRCTPASGGTSLHTPRSRPSSPGPSTWAGSQLSSCSVQVP (SEQ ID NO:96). Alsopreferred are polypeptide fragments having a biological activity asdescribed herein and the polynucleotides encoding the fragments.

The protein of the invention, cytogram, is a splice variant of GMP-17(or NKG7, GenBank accession number Q16617). Cytogram cDNA lacks thesecond exon of GMP-17 cDNA. Cytogram is a 142 amino-acid long protein,and GMP-17 is 165 amino acid long. The 53 amino-terminal amino acids areidentical between the two proteins, and the 89 carboxyl-terminal aminoacids are unique to cytogram. Cytogram displays a signal peptide(MELCRSLALLGGSLGLMFCLIALSTDFWFEAVGPTHS) (SEQ ID NO:97), a transmembranedomain (GDIISGHGPLVSTTAAFAAGK) (SEQ ID NO:98), and a PEC familymethallothionein domain(SCPCPQPSPWWWPWRCTPASGGTSLHTPRSRPSSPGPSTWAGSQLSSCSV) (SEQ ID NO:99) thatbinds to zinc ions.

Cytogram is a cytotoxic granule membrane protein that is constitutivelyand specifically expressed in NK cells (NKs) and cytotoxic T lymphocytes(CTLs). NK and CTL degranulation results in translocation of cytogramfrom granules to the plasma membrane. Cytogram contributes to theformation of junctions between effector cells and target cells followingexocytosis. Furthermore, once located on the plasma membrane, cytogramregulates ion channels required for cytotoxicity of NKs and CTLs andpromotes cytolytic effector function.

An embodiment of the invention is directed to a composition comprising acytogram polypeptide sequence of SEQ ID NO:42.

A further embodiment of the invention is directed to a compositioncomprising a cytogram polypeptide fragment having biological activity ofpromoting NK and CTL cytotoxicity.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:41 encoding a cytogrampolypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a cytogram polypeptidefragment having biological activity.

A further embodiment of the invention is directed to a compositioncomprising an antibody recognizing a cytogram polypeptide sequence ofSEQ ID NO:42 or a cytogram polypeptide fragment having biologicalactivity. Preferably, the antibody recognizes a non-linear epitope or anepitope located within the 89 C-terminal amino acids of cytogram.Preferably, the antibody binds to cytogram but not to GMP-17.Preferably, the antibody recognizes the AGKDSGLD (SEQ ID NO:100) aminoacid sequence.

A further embodiment of the present invention relates to a method ofbinding activated NKs and CTLs comprising the step of: contactingactivated NKs and CTLs with an antibody that specifically binds tocytogram polypeptides under conditions that allow binding of cytogrampolypeptides to the antibody. In a preferred embodiment, antibodies thatspecifically bind to cytogram polypeptides can be used to purifyactivated NKs and CTls. In such method, the antibody is preferablycovalently or not covalently attached to a solid matrix and allowed tobind cytogram polypeptides using techniques well known in the art. Thismethod comprises the steps of: i) contacting activated NKs and CTLs withan antibody that specifically binds to cytogram polypeptides underconditions that allow binding of cytogram polypeptides to the antibody,ii) washing the solid matrix to get rid of contaminants, and iii)eluting the cells of interest using more stringent conditions. Such amethod can for example be useful for purifying NKs and CTLs from cancerpatients prior to in vitro expansion.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a cytogram polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing cytogramexpression. Preferably, the polynucleotides capable of directingcytogram expression are located in the 5′ regulatory region of thecytogram gene. Further preferably, these polynucleotides are locatedwithin 500 base pairs of the cytogram coding region. Thesepolynucleotides preferably comprise a promoter sequence. Techniquesknown in the art for introducing polynucleotide sequences to endogenoussequences are described in U.S. Pat. No. 5,641,670 and PCT WO9629411,which disclosures are hereby incorporated by reference in theirentireties. Cytogram protein produced by said host cell may be used forin vitro detection and purification methods as well as diagnosis and invivo applications.

One embodiment of the present invention is directed to methods ofdetecting or quantifying activated NKs and CTLs comprising the steps of:i) contacting a body fluid, a tissue sample or a mammalian cell culturewith an antibody that specifically binds to cytogram polypeptides, andii) detecting the antibody in the sample using any detectable signal.Compounds such as, e.g., alkaline phosphatase, peroxidase, FITC,rhodamine, Texas-Red, biotin and digoxigenin can be used to provide adetectable signal. The detection of the signal may be carried out usingimmunohistological or immunofluorescing processes that are well-known tothose skilled in the art. The antibody that specifically binds tocytogram polypeptides may be directly labeled with the compound givingthe detectable signal. Alternatively, the antibody that specificallybinds to cytogram polypeptides is not labeled and detection of thesignal occurs indirectly by using labeled secondary antibodies. In apreferred embodiment, detecting activated NKs and CTLs can be used tomeasure the effect of a test compound on CTL or NK activity in mammaliancell cultures. In another preferred embodiment, such methods can be usedto monitor the effects of a treatment aiming to increase or decrease CTLor NK activity in a patient, or to detect the beginning of a graftrejection reaction in a patient.

An embodiment of the present invention relates to methods of usingcytogram or fragment thereof as a marker of cytotoxic granulescomprising the steps of: i) contacting a body fluid, a tissue sample ora mammalian cell culture with an antibody that specifically binds tocytogram polypeptides, and ii) detecting the localization of theantibody in the sample using any detectable signal. Compounds such as,e.g., FITC, rhodamine, Texas-Red, [³⁵S]methionine, [³⁵S]cysteine andbisbenzimide can be used to provide a detectable signal. The detectionof the signal may be carried out using immuno-electron microscopy orfluorescence microscopy. The antibody that specifically binds tocytogram polypeptides may be directly labeled with the compound givingthe detectable signal. Alternatively, the antibody that specificallybinds to cytogram polypeptides is not labeled and detection of thesignal occurs indirectly by using labeled secondary antibodies. Suchmethods of using cytogram or part thereof as a marker of cytotoxicgranules include those described by Medley et al. (Proc Natl Acad SciUSA. 93:685-9 (1996)), which disclosure is incorporated by reference inits entirety. CTLs and NKs form the major defense of higher organismsagainst virus-infected and transformed cells and a key function of CTLsis to detect and eliminate potentially harmful cells by lysis. Thus, amarker for detecting degranulation of NKs and CTLs is very useful whenscreening for drugs to treat or monitor the status or progression of,for example, infection with intracellular pathogens, graft versus hostdisease, susceptibility to transplantable and spontaneous malignancies,lymphoid homeostasis, the tendency to auto-immune diseases or whenstudying these diseases.

Another embodiment relates to a method of producing cytogrampolypeptides comprising the steps of: i) obtaining a cell capable ofexpressing a cytogram polypeptide; ii) growing said cell underconditions suitable to produce said polypeptide; and iii) purifying saidpolypeptide. The purification of the protein can be done following anytechnique well-known to those skilled in the art. Preferably, anantibody directed against cytogram or part thereof may be bound to achromatographic support to form an affinity chromatography column. Evenmore preferably, the antibody binds to cytogram but not to GMP-17. Thecell capable of expressing a cytogram polypeptide may be obtained by anyof the techniques well-known to those skilled in the art. A host cellmay be transfected with a recombinant expression vector comprising apolynucleotide of the present invention. Alternatively, a heterologouspromoter may be used. Preferably, the host cell is a mammalian hostcell.

Another preferred embodiment relates to a method of screening testsubstances for modulators of cytogram expression. This method comprisesthe steps of: i) contacting a cell with a test substance; and ii)comparing cytogram expression in the cell after exposure to the testsubstance to that of an unexposed control cell. Cytogram expression isdetermined by methods common to the art or included herein. Methods ofdetermining cytogram expression include but are not limited to methodsof quantifying cytogram polynucleotides (e.g., detection of cytogrammRNA by northern blot or RTPCR) or to methods of quantifying cytogrampolynucleotides (e.g., detection of cytogram proteins by western hot orimmunochemistry). Preferably, the test substance modifies the expressionof cytogram in a specific cell type while not in others. Mostpreferably, the test substance modifies cytogram expression specificallyin NKs and CTLs.

A further embodiment of the present invention is directed to testsubstances for modulators of cytogram activity and to a method ofscreening for such test substances comprising the steps of: i)contacting a cell with a test substance, ii) determining cytogramactivity, and iii) comparing cytogram activity in the cell afterexposure to that of an unexposed control cell. Cytogram activity can forexample be monitored by studying cytotoxic activity of NKs using thecytotoxic assay described in U.S. Pat. No. 5,229,494, which disclosureis hereby incorporated by reference in its entirety. Preferably,cytogram activity is studied in NKs or CTLs. Most preferably, asubstance that modulate cytogram activity is administered to anindividual in modulate NK or CTL cytotoxicity.

Test substances that decrease cytogram expression or activity aredefined as inhibitors or antagonists of cytogram. Test substances thatincrease cytogram expression or activity are defined as activators oragonists of cytogram. Tests substances that modulate the expression oractivity of cytogram include but are not limited to chemical compounds,oligonucleotides, ribozymes and antibodies. These substances may be madeand used according to methods well known in the art.

An embodiment of the present invention relates to a method of using thecompositions of the present invention to prevent or reduce in severitydisorders caused as a result of NK or CTL cytotoxicity. In a preferredembodiment, such a method comprises the step of administering to anindividual a cytogram antagonist in a physiologically acceptablecomposition. Preferably, such methods for reducing NK and CTL activationare directed to treat allergy and asthma, and to prevent on-going immuneresponses. More particularly, such methods can be used to prevent orreduce in severity graft rejections, graft versus host diseases andautoimmune diseases (e.g., rheumatoid arthritis, systemic lupus,multiple sclerosis, insulin-dependent diabetes, hepatitis, rheumatoidarthritis, Graves disease), and to induce tolerance to grafttransplantation (e.g., transplantation of cells, bone marrow, tissue,solid-organ, bone). A preferred cytogram antagonist for use in suchmethod is an antibody that specifically binds to cytogram polypeptides.

In another preferred embodiment, an anti-cytogram antibody may be usedas a targeting and delivery mechanism for bringing a pharmaceuticalagent to activated NKs and CTLs. Such a method comprises the steps of:i) conjugating an anti-cytogram antibody to a pharmaceutical agent; andii) administering a composition comprising said conjugate and apharmaceutically acceptable carrier to an individual suffering from adisorder caused as a result of NK or CTL cytotoxicity. Preferably, ananti-cytogram antibody is used in an antibody-directed enzyme-prodrugtherapy comprising the steps of: i) conjugating an anti-cytogramantibody to an enzyme converting a relatively non-toxic compound into asubstantially more toxic compound; and ii) administering said relativelynon-toxic compound to a patient after a delay that allowed residualenzyme-antibody conjugate to be cleared from the blood. Such a methodmay be performed as described by Melton et al. (J. Natl. Cancer Inst.1996 88:153-65 (1996)), which disclosure is hereby incorporated byreference in its entirety.

An embodiment of the present invention relates to methods of using thecompositions of the present invention to enhance NK or CTL cytotoxicity.A preferred method directed to enhance NK or CTL cytotoxicity comprisesthe step of introducing a cytogram polypeptide or a cytogram agonist ina physiologically acceptable carrier in an individual suffering fromdiseases and disorders where a boost to the immune system is desirable.More particularly, such treatments can be used in conjunction with aradiation therapy or a chemotherapy when treating a cancer, and can beused to treat neoplastic disorders (e.g., multiple myeloma, coloncancer, hepatoma) viral infections (e.g., HIV, HBV, HCV, hepatitis,measles and herpes viruses infections), and various immune deficiencies.These immune deficiencies may be genetic (e.g. severe combinedimmunodeficiency (SCID)) or be caused by various bacterial or fungalinfections (e.g. infections by mycobacteria, Leishmania spp., malariaspp. and candidiasis).

Physiologically acceptable carriers can be prepared by any method knownby those skilled in the art. Physiologically acceptable carriers includebut are not limited to those described in Remington's PharinaceuticalSciences (Mack Publishing Company, Easton, USA 1985), which disclosureis hereby incorporated by reference in its entirety. Pharmaceuticalcompositions can be for, e.g., intravenous, topical, rectal, local,inhalant or subcutaneous, intradermal, intramuscular, oral andintracerebral use. The compositions can be in liquid (e.g., solutions,suspensions), solid (e.g., pills, tablets, suppositories) or semisolid(e.g., creams, gels) form. Dosages to be administered depend onindividual needs, on the desired effect and the chosen route ofadministration.

Another embodiment is directed to a method of using cytogrampolynucleotides comprising the steps of: i) constructing a recombinantmolecule comprising a nucleic acid sequence encoding a cytogrampolypeptide that allows expression of cytogram or part thereof undergiven physiological conditions, and ii) introducing this recombinantmolecules into a cell, a mammal or a human. Such a method can be used totreat, e.g., the genetic immune deficiencies and neoplastic diseaseslisted above. Preferably, the recombinant molecule is introduced intoNKs or CTLs.

Still another embodiment is directed to a method of using antisensecytogram polynucleotides comprising the steps of: i) constructing arecombinant molecule comprising a nucleic acid sequence encoding a cDNAthat is complementary to cytogram polynucleotides, and ii) introducingthis recombinant molecules into a cell, a mammal or a human. Such amethod can be used to treat autoimmune diseases. Alternatively, such amethod can be used to prevent the autoimmune disorders listed above,graft versus host disease or graft rejection when transplanting anorgan. The recombinant molecule may be introduced into NKs, CTLs, orgraft cells.

Recombinant molecules comprising a nucleic acid sequence encoding acytogram polypeptide or a nucleic acid sequence that encodes a cDNA thatis complementary to cytogram polynucleotides may be directly introducedinto cells or tissues in vivo using delivery vehicles such as retroviralvectors, adenoviral vectors and DNA virus vectors. They may also beintroduced into cells in vivo using physiological techniques such asmicroinjection and electroporation or chemical methods such ascoprecipitation and incorporation of DNA into liposomes. Recombinantmolecules may also be delivered in the form of an aerosol or by lavage.The cytogram polynucleotides may also be applied extracellularly such asby direct injection into cells. Preferably, cytogram polynucleotides areintroduced into NKs or CTLs.

Protein of SEQ ID NO:44 (Internal Designation Clone 1000838982220-20-4-0-C2-F)

The cDNA of Clone 1000838982_(—)220-20-4-0-C2-F (SEQ ID NO: 43) encodesTetranab of SEQ ID NO:44, comprising the amino acid sequence:MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRASWSEACGTS. Accordingly,it will be appreciated that all characteristics and uses of thepolypeptides of SEQ ID NO:44 described throughout the presentapplication also pertain to the polypeptides encoded by the human cDNAincluded in Clone 1000838982_(—)220-20-4-0-C2-F. In addition, it will beappreciated that all characteristics and uses of the polynucleotides ofSEQ ID NOs:43 described throughout the present application also pertainto the nucleic acids comprising the human cDNA included in Clone1000838982_(—)220-20-4-0-C2-F. A preferred embodiment of the inventionis directed toward the compositions of SEQ ID NO:44, SEQ ID NO:43, andClone 1000838982_(—)220-20-4-0-C2-F. Also preferred are polypeptidefragments having a biological activity as described herein and thepolynucleotides encoding the fragments.

The protein of the invention, Tetranab, is a variant of CD37 (GenBankaccession number P11049). CD37 is a molecular facilitator that bringstogether molecular complexes necessary for T-cell dependant B-cellresponse. CD37 stabilizes molecular interactions, resulting in a moreefficient response. Tetranab is a divergent member of the tetraspaninfamily, which displays one signal anchor (FNLFFFVLGSLIFCFGIWILI) (SEQ IDNO:101) and two transmembrane domains (VLAISGIFTMGIALLGCVGAL (SEQ IDNO:102) and LYFGMLLLLFATQITLGILIS (SEQ ID NO:103)). Tetranab isspecifically expressed on mature B cells. It is not expressed duringdifferentiation of B cells and its expression is down-regulated withactivation of B cells. Tetranab acts as a dominant negative inhibitor ofCD37-facilitated assembly of functional complexes at B cells surfaces.Thus Tetranab prevents CD37-dependent activation of B cells.

An embodiment of the invention is directed to a composition comprising aTetranab polypeptide sequence of SEQ ID NO:44.

A further embodiment of the invention is directed to a compositioncomprising a Tetranab polypeptide fragment having biological activity ofpreventing T-cell dependant B-cell response.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:43 encoding a Tetranabpolypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a Tetranab polypeptidefragment having biological activity of preventing T-cell dependentB-cell response.

A further embodiment of the invention is directed to a compositioncomprising an antibody recognizing a Tetranab polypeptide. Preferably,the antibody recognizes an epitope comprising one or more of the 9C-terminal amino acids of Tetranab, wherein one or more of the 9C-terminal amino acids is required for antibody binding. Preferably, theantibody binds specifically to Tetranab but not to CD37. As used herein,an anti-Tetranab antibody refers to an Tetranab-specific antibody or anantigen binding fragment thereof.

The present invention also relates to a method of binding such anantibody to a Tetranab polypeptide comprising the step of: contacting aTetranab polypeptide with said antibody or an antigen-binding fragmentthereof under conditions that allow binding to take place. Suchconditions are well known to those skilled in the art. Such methods areuseful for detecting Tetranab polypeptides or for purifying B cells asfurther described herein.

An embodiment of the present invention is directed to a method ofdetecting Tetranab polypeptides in a biological sample, said methodcomprising the steps of: i) contacting a biological sample with ananti-Tetranab antibody; and ii) detecting the antigen-antibody complexformed. The antibody or antibody fragment may be monoclonal orpolyclonal. In addition, the antibody or antibody fragment may beprimarily or secondarily labeled by any detectable compound (e.g.,radioactive, fluorescent, luminescent, or enzymatic) common in the art.Further preferred is a method of using said anti-Tetranab antibodies orantigen binding fragments in a flow cytometric analysis of Tetranabexpression by B cells. Such a method can be used as a diagnostic toolfor detecting the presence of a Tetranab polypeptide as furtherdescribed herein.

Another embodiment of the present the invention is directed to adiagnostic kit for detecting in vitro the presence of a Tetranabpolypeptide. Such kit comprises: i) a polyclonal or monoclonal antibodyor fragment thereof that specifically binds a Tetranab polypeptide; andoptionally, ii) a reagent allowing the detection of the antigen-antibodycomplexes formed. Preferably, the antibody or antibody fragment isdetectably labeled as described above The optional reagent may provide adetectable signal, and either bind to the antibody, or react with thelabel on said antibody. Preferably, the kit comprising anti-Tetranabantibodies or antigen binding fragments is used for diagnosingnon-Hodgkin's lymphomas, or for monitoring the condition of a patientreceiving treatment for a non-Hodgkin's lymphoma. Optionally, said kitfor diagnosing non-Hodgkin's lymphomas comprises a sample of anindividual suffering from lymphoma, as well as a control sample from anormal individual.

Another preferred embodiment relates to a method for detecting in vivothe presence of a Tetranab polypeptide using anti-Tetranab antibodies.Such a method is very useful for imaging of tumor involved sites, andespecially for imaging of non-Hodgkin's lymphomas. Preferably, imagingis carried out using ¹¹¹In or ¹³¹I conjugated to an anti-Tetranabantibody as described by Bunn et al (Lancet 2:1219-21 (1894)), whichdisclosure is incorporated by reference in its entirety. This embodimentalso relates to a kit for use in tumor imaging such as the kit describedabove.

Still another embodiment of the present invention relates to a methodusing an anti-Tetranab antibody for immunotherapeutic purposescomprising the step of administering a therapeutic dose of aradioactively labeled anti-Tetranab antibody in a pharmaceuticallyacceptable carrier. Such a method can be applied to treat non-Hodgkin'slymphomas and post-transplant lymphoproliferative disorders. In such amethod, labeling with ⁹⁰Y or ¹³¹I is preferred. Radioactive isotopes canbe attached to the anti-Tetranab antibody by a number of methodswell-known to those skilled in the art, e.g., by covalent attachment ofa iodine isotope directly to the antibody or by covalent attachment of achelating moiety to the antibody allowing the chelator to coordinate themetal isotope. Routes of administration may vary widely. Preferred modesof administration are intravenous injection and intralymphatic routes ofadministration such as subcutaneous and intramuscular injection, orcatherization of lymphatic vessels. The radiometric dosage to be appliedcan vary substantially. Methods of administration and dosage regimen fortreating lymphomas can be determined as described in U.S. Pat. No.5,595,721, which disclosure is hereby incorporated by reference in itsentirety.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a Tetranab polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing Tetranabexpression. Preferably, the polynucleotides capable of directingTetranab expression are located in the 5′ regulatory region of theTetranab gene. Further preferably, these polynucleotides are locatedwithin 500 base pairs of the Tetranab coding region. Thesepolynucleotides preferably comprise a promoter sequence. Techniquesknown in the art for introducing polynucleotide sequences to endogenoussequences are described in U.S. Pat. No. 5,641,670 and PCT WO9629411,which disclosures are hereby incorporated by reference in theirentireties. Tetranab protein produced by said host cell may be used forin vitro detection and purification methods as well as diagnosis and invivo applications.

An embodiment of the invention provides for a method of screening testsubstances for modulators of Tetranab expression. This method comprisesthe steps of: i) contacting a cell with a test substance; and ii)comparing Tetranab expression in the cell after exposure to the testsubstance to that of an unexposed control cell. Tetranab expression isdetermined by methods common to the art or included herein, by detectingTetranab polynucleotides or polypeptides. An example of this methodcomprises the steps of: i) culturing two equivalent cell samples; ii)adding a test substance to one of the cultures and not the other; iii)harvesting both cultures at a specified time; iv) purifying the mRNAfrom each sample of cells; v) comparing the level of Tetranab mRNA ineach sample by Northern blot, RTPCR, or another method common to theart. An additional example comprises the steps of: i) having twoequivalent cultures of cells; ii) adding a test substance to one of thecultures and not the other; iii) harvesting both cultures; iv) purifyingthe protein from each sample of cells; v) comparing the level ofTetranab polypeptides in each sample by Western blot,immunohistochemistry, or another method common to the art. Preferably,Tetranab expression is measured in B cells.

In another embodiment, a Tetranab polypeptide or a fragment thereof maybe used to screen for compounds that modulate Tetranab activity. Thismethod comprises the steps of: i) contacting a Tetranab polypeptide orfragment thereof with a test substance, and ii) monitoring Tetranabactivity. Tetranab activity may be determined by monitoring B cellactivation. For example, B cell function can be determined by measuringcalcium ion flux across the membrane as described in U.S. Pat. No.4,987,084, which disclosure is incorporated by reference in itsentirety.

Test substances that decrease Tetranab expression or activity aredefined as inhibitors or antagonists of Tetranab. Test substances thatincrease Tetranab expression or activity are defined as activators oragonists. Agents that modulate the expression or activity of Tetranabactivity of the subject invention include, but are not limited toantisense oligonucleotides, ribozymes, and antibodies. These agents maybe made and used according to methods well known in the art.

A further embodiment of the present invention relates to a method ofantagonizing CD37 action, thus preventing T-cell dependant B-cellactivation. Such a method comprises the step of contacting a B cell witha composition comprising a Tetranab agonist. The B cells can be treatedin vitro or in vivo by directly administering the composition of thepresent invention to the cells. In a preferred embodiment, thisinvention provides a method of inhibiting B cell activation in anindividual comprising the step of administering to the individual aneffective inhibiting amount of a pharmaceutical composition comprising aTetranab agonist and a pharmaceutically acceptable carrier. A method ofinhibiting B cell activation is a useful method for inhibiting theimmune response of an individual. Preferably, inhibiting the immuneresponse using a method of the present invention is directed to inhibita graft rejection reaction, to treat an individual suffering fromautoimmune disease (e.g., rheumatoid arthritis, Myasthenia gravis,systemic lupus erythematosus, Graves' disease, idiopathichrombocytopenia purpura, hemolytic anemia, diabetes mellitus anddrug-induced autoimmune diseases such as drug-induced lupus), or toreduce allergic responses (e.g., asthma, hay fever and allergy topenicillin).

Another embodiment of the present invention relates to a method offavoring CD37 action, thus stabilizing molecular complexes that arenecessary for T cell dependant B cell activation. Such a methodcomprises the step of contacting a B cell with a composition comprisinga Tetranab antagonist. In this aspect of the invention, an effectiveinhibiting amount of a pharmaceutical composition comprising a Tetranabantagonist and a pharmaceutically acceptable carrier may be administeredto an individual in order to enhance B cell activation. Such a method isuseful for treating disorders where a boost of the immune system isdesirable such as immune deficiencies (e.g., severe combinedimmunodeficiency), various viral, fungal or bacterial infections (e.g.,HBV, HCV, HIV, hepatitis, measles and herpes virus infections,mycobacteria, Leishmania spp., malaria spp. and candidiasis infections),and in association with a radiation or chemotherapy.

Compositions comprising a Tetranab agonist or a Tetranab antagonist maybe formulated using a variety of acceptable excipients known in the art.Typically, the compositions are administered topically, orally or byinjection, either intravenously or intraperitoneally. Methods toaccomplish this administration and to determine the dosage regimen areknown to those of ordinary skill in the art.

Another embodiment relates to a method of producing Tetranabpolypeptides comprising the steps of: i) transfecting a host cell with arecombinant expression vector comprising a polynucleotide of the presentinvention, and ii) purifying the produced protein. The purification ofthe protein can be done following any technique well-known to thoseskilled in the art. Preferably, an antibody directed against Tetranab orpart thereof may be bound to a chromatographic support to form anaffinity chromatography column. Such a purified Tetranab polypeptide mayfor example be useful for preparing the pharmaceutical compositionsdescribed above.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a Tetranab polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing Tetranabexpression. Preferably, the polynucleotides capable of directingTetranab expression are located in the 5′ regulatory region of theTetranab gene. Further preferably, these polynucleotides are locatedwithin 500 base pairs of the Tetranab coding region. Thesepolynucleotides preferably comprise a promoter sequence. Techniquesknown in the art for introducing polynucleotide sequences to endogenoussequences are described in U.S. Pat. No. 5,641,670 and PCT WO9629411,which disclosures are hereby incorporated by reference in theirentireties. In preferred embodiments, the recombinant molecule isintroduced into an Escherichia coli cell or into a human B cell.Alternatively, the recombinant molecule is a vector comprising anheterologous promotor that increases Tetranab expression that isintroduced into a human cell. In a preferred embodiment, saidrecombinant molecule is introduced into animals, more particularlymammals. Said recombinant molecule may be introduced into animals inseveral ways. A preferred method of introducing the recombinant moleculeinto animals comprises the steps of: i) introducing the recombinantmolecule into a host cell by means known in the art such as, e.g.,transformation, electroporation, lipofection, microinjection, andtransduction, including the use of retroviral vectors, adenoviralvectors and DNA virus vectors; and ii) administering the recombinantcells to an animal. For example, the cells can be administered byinfusion. Such a method may be directed to treat, e.g., autoimmunediseases. Alternatively, the recombinant molecule may encode a cDNA thatis complementary to SEQ ID NO:43 or part thereof, and the method may beused to treat, e.g., immune deficiencies.

In another preferred embodiment, said recombinant molecule is used toestablish transgenic model animals by any method familiar to thoseskilled in the art. For example, transgenic mice may be established asdescribed by Hogan et al. (“Manipulating the Mouse Embryo: A LaboratoryManual”, Cold Spring Harbor Laboratory), which disclosure isincorporated herein by reference. By modulating in vivo the expressionof the transgene with drugs or modifier genes (activator or suppressorgenes), animal models can be developed that mimic human disorders suchas those listed above. These animal models thus allow the identificationof potential therapeutic agents for treatment of the disorders. Inaddition, recombinant cell lines derived from these transgenic animalsmay be used for similar approaches ex vivo.

Protein of SEQ ID NO:46 (Internal Designation Clone 500720840205-20-1-0-B7-F)

The cDNA of Clone 500720840_(—)205-20-1-0-B7-F (SEQ ID NO:45) encodesPDI protein of SEQ ID NO:46 comprising the amino acid sequence:MRLRRLALFPGVALLLAAARLAAASDVLELTDDNFESRISDTGSAGLMLVEFFAPWCGHCKRLAPEYEAAATRLKGIVPLAKVDCTANTNTCNKYGVSGYPTLKIFRDGEEAGAYDGPRTADGIVSHLKKQAGPASVPLRTEEEFKKFISDKDASIVGFFDDSFSEAHSEFLKAASNLRDNYRFAHTNVESLVNEYDDNGEGIILFRPSHLTNKFEDKTVAYTEQKMTSGKIKKFIQENIFGICPHMTEDNKDLIQGKDLLIAYYDVDYEKNAKGSNYRRNRVMMVAKKFLDAGHKLNFAVASRKTFSHELSDFGLESTAGEIPVVAIRTAKGEKFVMQEEFSRDGKALERFLQDYFDGNLKRYLKSEPIPESNDGPVKVVVAENFDEIVNNENKDVLIEFYAPWCGHCKNLEPKYKELGEKLSKDPNIVIAKMDATANDVPSPYEVRGFPTIYFSPANKKLNPKKYEGGRELSDFISYLQREATIPPVIQEEKPKKKKKAQEDL. Accordingly, it will be appreciated that allcharacteristics and uses of the polypeptides of SEQ ID NO:46 describedthroughout the present application also pertain to the polypeptidesencoded by the human cDNA included in Clone500720840_(—)205-20-1-0-B7-F. In addition, it will be appreciated thatall characteristics and uses of the polynucleotides of SEQ ID NO:45described throughout the present application also pertain to the nucleicacids comprising the human cDNA in Clone 500720840_(—)205-20-1-0-B7-F. Apreferred embodiment of the invention is directed toward thecompositions of SEQ ID NO:45, SEQ ID NO:46 and Clone500720840_(—)205-20-1-0-B7-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

The protein of SEQ ID NO:46, PDI, is a polymorphism variant of proteindisulfide isomerase A3 precursor (accession numbers U42068). The proteinof the invention displays two thioredoxin domain:ASDVLELTDDNFESRISDTGSAGLMLVEFFAPWCGHCKRLAPEYEAAATRLKGIVPLAKVDCTANTNTCNKYGVSGYPTLKIFRDGEEAGAYDGPRTADGIVSHLKKQAG (SEQ ID NO:104); andDGPVKVVVAENFDEIVNNENKDVLIEFYAPWCGHCKNLEPKYKELGEKLSKDPNIVIAKMDATANDVPSPYEVRGFPTIYFSPANKKLNPKKYEGGRELSDFISYLQREAT (SEQ ID NO:105). Also,the 505 amino acid protein of PDI displays one membrane-spanningsegment: SDTGSAGLMLVEFFAPWCGHC (SEQ ID NO:106).

Thioredoxin family active site proteins are a superfamily of proteinsthat participate in redox reactions and are distributed among a widerange of living organisms. The reduced form of thioredoxin is known toactivate some enzymes by reducing disulfide bridges that control theiractivity. In addition, thioredoxin is an electron donor in the reactionssequence that reduces ribonucleotides to deoxyribonucleotides catalyzedby ribonucleotide reductase. It has been reported that in humans,thioredoxin and the cellular redox state modified by thioredoxin play acrucial role in arterial neointima formation in atheroscleriosis.Thioredoxin is involved in cellular defense mechanisms against oxidativedamage. Thioredoxin has also been implicated in regulatingglucocorticoid responsiveness to cellular oxidative stress responsepathways. In particular, thioredoxin is capable of sensing andtransmitting the redox state of the cell to the glucorticoid receptor bytargeting both the ligand- and DNA-binding domains of the receptor.Human thioredoxin has been suggested to act as a free radical scavengerand has been shown to limit the extent of ischemia reperfusion injury.Multiple in vitro substrates for thioredoxin have been identified,including ribonuclease, choriogonadotropins, coagulation factors,glucocorticoid receptor, and insulin.

PDI belongs to the protein disulfide isomerase class of thioredoxinfamily active site-containing proteins that catalyze the oxidation ofthiols, reduction of disulfide bonds, and isomerization of disulfides,depending on the reaction conditions. PDI catalyzes the formation ofcorrect disulfide pairing in nascent proteins. PDI preferentiallyinteracts with peptides that contain cysteine residues but is otherwiseundiscriminating. The broad substrate specificity of PDI enables it tospeed the folding of diverse disulfide-containing proteins. By shufflingdisulfide bonds, PDI enables proteins to quickly find the mostthermodynamically stable pairings amongst available cysteine residues.Consequently, PDI is involved in protein processing, protein folding,and protein secretion. PDI is also involved in collagen andcollagen-like protein biosynthesis and mutations of PDI causeEhlers-Danlos syndrome.

An embodiment of the invention is directed to a composition comprising aPDI polypeptide sequence of SEQ ID NO:46.

A further embodiment of the invention is directed to a compositioncomprising a PDI polypeptide fragment having a biological activity ofbinding peptides with cysteine residues.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:45 encoding a PDIpolypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a PDI polypeptide fragmenthaving biological activity of binding peptides with cysteine residues.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a PDI polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing PDI expression.Preferably, the polynucleotides capable of directing PDI expression arelocated in the 5′ regulatory region of the PDI gene. Further preferably,these polynucleotides are located within 500 base pairs of the PDIcoding region. These polynucleotides preferably comprise a promotersequence. Techniques known in the art for introducing polynucleotidesequences to endogenous sequences are described in U.S. Pat. No.5,641,670 and PCT WO9629411, which disclosures are hereby incorporatedby reference in their entireties. PDI protein produced by said host cellmay be used for in vitro detection and purification methods as well asdiagnosis and in vivo applications.

A method of oxidating thiols species comprising the step of: contactinga PDI polypeptide with peptides with reduced cysteine residues.Preferably, thiols are oxidized to disulfide bonds. More preferably, thedisulfide bonds are correctly isomerized. This method may be applied torecombinant production of protein sequences containingcollagen/collagen-like protein sequences.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a PDI polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing PDI expression.Preferably, the polynucleotides capable of directing PDI expression arelocated in the 5′ regulatory region of the PDI gene. Further preferably,these polynucleotides are located within 500 base pairs of the PDIcoding region. These polynucleotides preferably comprise a promotersequence.

An embodiment of the present invention relates to compositionscomprising PDI polypeptides. The method of producing PDI polypeptidescomprises the steps of: i) transfecting a mammalian host cell with arecombinant expression vector comprising a polynucleotide of the presentinvention, and ii) purifying the produced protein. The purification ofthe protein can be done following any technique well known to thoseskilled in the art. Preferably, an antibody directed against PDI or partthereof, preferably, an antibody directed against the Thioredoxin domainof the polypeptide, may be bound to a chromatographic support to form anaffinity chromatography column.

Another embodiment of the invention provides for a method utilizing PDIalone or in combination with a reductant or reduction system to reduceprotein intramolecular disulfide bonds. For example, PDI may be used incombination with glutenins or gliadins present in flour or seeds toimprove dough strength and baked goods characteristics such as crumbquality, softness, and higher loaf volume as further described in U.S.Pat. No. 6,113,951, which disclosure is hereby incorporated in itsentirety.

Further preferred is a method of reducing a toxic protein having one ormore intramolecular cystines comprising contacting the toxic proteinwith an amount of PDI effective for reducing the protein, andmaintaining the contact for a time sufficient to reduce one or moredisulfide bridge and denature the toxic protein (as further described inU.S. Pat. No. 6,113,951, which disclosure is hereby incorporated in itsentirety).

In one embodiment, PDI may be co-expressed with a desired protein orproteins in a host cell which is used to produce the desired protein orproteins. The co-expression of PDI will result in an increase in theamount of correctly folded protein obtained from the host cell. In thismethod, a first vector expressing PDI and a second vector expressing adesired protein are introduced into a host cell using conventionalmethods, and following expression, the desired protein is harvested.

In another embodiment, PDI may be added to a protein sample duringprocessing to increase the efficiency of protein processing. Processingof protein will be carried out following expression of at least onedesired protein in the host cell and may include purification,renaturation, solubilization or any other steps used to obtain a pureand active form of a desired protein. In this embodiment, PDI may beadded during these steps to minimize loss of protein resulting, forexample, from protein oxidation, aggregation, or improper folding. Theamount of PDI to be added may be determined by one of skill usingroutine methods, and will be sufficient to reduce the loss of protein.

In another embodiment, the invention provides a method for neutralizingfood allergens by PDI as described in U.S. Pat. No. 6,190,723, whichdisclosure is hereby incorporated by reference in its entirety. A methodof decreasing the allergenicity of an allergenic food protein comprisesthe step of: contacting the protein with and amount of PDI, Nicotinamideadenine dinucleotide phosphate-thioredoxin reductase and NADPH, or anamount of PDI and dithiothreitol effective for decreasing theallergenicity of the protein.

In another embodiment, the invention provides a method for increasingthe digestibility of food proteins. This method may be carried out asdescribed in U.S. Pat. No. 5,952,034, which disclosure is herebyincorporated by reference in its entirety. A method of increasing thedigestibility of a food protein comprises the step of: treating a foodwith an amount of PDI, nicotinamide adenine dinucleotidephosphate-thioredoxin reductase (NTR) and NADPH effective for increasingthe digestibility of the food.

An embodiment of the invention provides for a method of screening testsubstances for modulators of PDI expression. This method comprises thesteps of: i) contacting a cell with a test substance; and ii) comparingPDI expression in the cell after exposure to the test substance to thatof an unexposed control cell. PDI expression is determined by methodscommon to the art or included herein, by detecting PDI polynucleotidesor polypeptides. An example of this method comprises the steps of: i)culturing two equivalent cell samples; ii) adding a test substance toone of the cultures and not the other; iii) harvesting both cultures ata specified time; iv) purifying the mRNA from each sample of cells; v)comparing the level of PDI mRNA in each sample by Northern blot, RTPCR,or another method common to the art. The invention provides for designand use of specific polynucleotide probes and primers, as discussedherein. An additional example comprises the steps of: i) having twoequivalent cultures of cells; ii) adding a test substance to one of thecultures and not the other; iii) harvesting both cultures; iv) purifyingthe protein from each sample of cells; v) comparing the level of PDIpolypeptides in each sample by Western blot, immunohistochemistry, oranother method common to the art. The invention provides for design anduse of specific antibodies and antibody fragments, as discussed herein.This method may be applied to diagnosis of PDI-related disorders. Forexample, an abnormally high level of PDI indicates presence of primarytumors such as lung, colon, cervical and hepatocellular carcinoma.

Over-expression of PDI plays a role in the development of disease.Increased expression of PDI is associated with Sjogrens' syndrome,increased growth of leukemia and lymphoma cells, and the proliferationof virally transformed cells. A deficiency of PDI is associated withimmunological diseases, including, but not limited to, atherosclerosis,stroke, asthma, allergies, Crohns' disease, ulcerative colitis,diabetes, Hermansky-Pudlack syndrome, Alzheimers disease, and damage totissues caused by trauma, ischemia, hypoxia, radiation and ultravioletexposure.

In another embodiment, a PDI polypeptide or a fragment thereof, may beused to screen for compounds that activate or inhibit PDI activity. Thismethod comprises the steps of: i) contacting a PDI polypeptide orfragment thereof with a test substance and ii) monitoring PDI activity.

Test substances that decrease PDI expression or activity are defined asinhibitors or antagonists of PDI. Test substances that increase PDIexpression or activity are defined as activators or agonists. Inhibitorsof PDI include, but are not limited to antisense oligonucleotides,ribozymes, and antibodies. These agents may be made and used accordingto methods well known in the art. Disorders characterized by aberrantPDI expression is a cardiovascular disorder, e.g. atherosclerosis,ischaemia reperfusion injury, cardiac hypertrophy, hypertension,coronary artery disease, myocardial infarction, arrhythmia,cardiomyopathies, and congestive heart failure; a connective tissuedisorder, e.g., Ehlers-Danlos Syndrome; or a hepatic disorder, e.g.,alcoholic liver disease, liver cirrhosis and liver cancer.

In another embodiment, the protein of the present invention, as well asagents, or modulators which have a stimulatory or inhibitory effect onPDI activity as identified by a screening assay can be administered toindividuals to treat (prophylactically or therapeutically) disorders,described herein, associated with aberrant PDI activity.

In another embodiment of the invention, PDI, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, between PDIand the agent being tested, may be measured. Another technique for drugscreening which may be used provides for high throughput screening ofcompounds having suitable binding affinity to the protein of interest asdescribed in published PCT application WO84/03564, which disclosure ishereby included in its entirety. In this method, as applied to PDI,large numbers of different small test compounds are synthesized on asolid substrate, such as plastic pins or some other surface. The testcompounds are reacted with PDI, or fragments thereof, and washed. BoundPDI is then detected by methods well known in the art. Purified PDI canalso be coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding PDI specificallycompete with a test compound for binding PDI. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with PDI.

In one embodiment, PDI or a biologically active fragment thereof isadministered to a subject to treat conditions or diseases associatedwith a deficiency of PDI. These include, but are not limited to, cancersof the brain, prostate, breast, bladder, and thyroid, and immunologicaland infectious diseases, including, but not limited to, atherosclerosis,stroke, asthma, allergies, Crohns' disease, ulcerative colitis,diabetes, Hermansky-Pudlack syndrome, Alzheimers disease, and damage totissues caused by trauma, ischemia, hypoxia, radiation and ultravioletexposure.

In another embodiment, a vector capable of expressing PDI, or a fragmentor a derivative thereof, may also be administered to a subject to treator prevent diseases or conditions described above.

In one embodiment, agonists of PDI may be administered to a subject totreat or prevent diseases or conditions described above.

In another embodiment, a vector expressing antisense of thepolynucleotide encoding PDI may be administered to a subject to treat orprevent conditions or diseases associated with over-expression of PDI.Such conditions or diseases include lymphoma, leukemia, Sjogrens'syndrome, human T-lymphotropic virus, Epstein-Barr virus, humanimmunodeficiency virus. An example is described in published PCTapplication WO 99/38963, which disclosure is hereby included in itsentirety.

Another embodiment of the invention relates to composition and methodsusing polynucleotide sequences encoding the protein of the invention orpart thereof to establish transgenic model animals (D. melanogaster, M.musculus), by any method familiar to those skilled in the art. Bymodulating in vivo the expression of the transgene with drugs ormodifier genes (activator or suppressor genes), animal models can bedeveloped that mimic human hormone-dependent disorders such as cancers.These animal models would thus allow the identification of potentialtherapeutic agents for treatment of the disorders. In addition,recombinant cell lines derived from these transgenic animals may be usedfor similar approaches ex vivo.

In another embodiment, an array of oligonucleotides probes comprisingthe nucleotide sequence of PDI or fragments thereof can be constructedto conduct efficient screening of e.g., genetic mutations or deletion.The microarray can be used to monitor the expression level of largenumbers of genes simultaneously and to identify genetic variants,mutations, and polymorphisms. This information may be used to determinegene function, to understand the genetic basis of a disorder, todiagnose a disorder, and to develop and monitor the activities oftherapeutic agents (see for example: Chee, M. et al., Science,274:610-614 (1996) which disclosure is hereby incorporated by referencein its entirety). For example, mutations of PDI cause Ehlers-Danlossyndrome.

Protein of SEQ ID NO:48 (Internal Designation Clone 146821106-020-1-0-G3-F)

The cDNA of Clone 146821_(—)106-020-1-0-G3-F (SEQ ID NO:47) encodesNBART protein of SEQ ID NO:48, comprising the amino acid sequence:MLVMYLLAALFGYLTFYGEVEDELLHAYSKVYTLDIPLLMVRLAVLVAVTLTVPIVLFPIRTSVITLLFPKRPFSWIRHFLIAAVLIALNNVLVILVPTIKYIFGFIGASSATMLIFILPAVFYLKLVKKETFRSPQKVGALIFLVVGIFFMIGSMALIIIDWIYDPPNSKHH. Accordingly, it will beappreciated that all characteristics and uses of the polypeptides of SEQID NO:48 described throughout the present application also pertain tothe polypeptides encoded by the human cDNA included in Clone146821_(—)106-020-1-0-G3-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:47described throughout the present application also pertain to the nucleicacids comprising the human cDNA in Clone 146821_(—)106-020-1-0-G3-F. Apreferred embodiment of the invention is directed toward thecompositions of SEQ ID NO:47, SEQ ID NO:48 and Clone146821_(—)106-020-1-0-G3-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

ADP ribosylation factor (ARF) and ARF-like (ARL) proteins comprise theARF family within the Ras superfamily of regulatory GTPases. Members ofthis superfamily function as molecular nodes in signaling that candirectly activate one or more enzymatic activities or coordinate therecruitment and assembly of more elaborate multi-subunit complexes. ARL2is a member of a functionally distinct group of ARF-like genes. ARL2binds GTP rapidly and hydrolyzes it. In the GDP-bound form, ARL2interacts with the tubulin-specific chaperone cofactor D. Thisinteraction prevents the destruction of tubulin and microtubles byoverexpressed cofactor D. The tubulin GTPase activating protein activityof cofactor D is also inhibited by ARL2 binding. This interactionprevents ARL2 contributes to modulating microtubule dynamics. NBARTbinds specifically to ARL2-GTP with high affinity but does not interactwith ARL2-GDP.

An embodiment of the invention is directed to a composition comprising aNBART polypeptide sequence of SEQ ID NO:48.

A further embodiment of the invention is directed to a compositioncomprising a NBART polypeptide fragment having a biological activity ofbinding to ARL2.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:47 encoding a NBARTpolypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a NBART polypeptidefragment having biological activity of binding to ARL2.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a NBART polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing NBART expression.Preferably, the polynucleotides capable of directing NBART expressionare located in the 5′ regulatory region of the NBART gene. Furtherpreferably, these polynucleotides are located within 500 base pairs ofthe NBART coding region. These polynucleotides preferably comprise apromoter sequence. Techniques known in the art for introducingpolynucleotide sequences to endogenous sequences are described in U.S.Pat. No. 5,641,670 and PCT WO9629411, which disclosures are herebyincorporated by reference in their entireties. NBART protein produced bysaid host cell may be used for in vitro detection and purificationmethods as well as diagnosis and in vivo applications.

A preferred embodiment of the invention is a method of using NBART tobind ARL2. This method comprises the step of contacting a NBARTpolypeptide or active fragment thereof with an ARL2 protein underconditions that allow NBART binding, whereby binding inhibits theactivity of ARL2.

A preferred embodiment of the invention is a method of purifying ARL2.This method comprises the steps of: contacting a NBART polypeptide ofactive fragment thereof with a ARL2 under conditions that allow binding;removing contaminants; and eluting the ARL2 with more stringentconditions. Preferably, the NBART peptide is immobilized on a solid orsemi-solid matrix to facilitate washing of sample to removecontaminants.

A preferred embodiment of the inventions is a method of detecting ARL2.This method comprises the step of contacting a NBART polypeptide oractive fragment thereof with ARL2 and detecting the presence of saidARL2 by detecting NBART. Preferably, the NBART polypeptide is detectablylabeled with, for example, a fluorescent, luminescent, or radioactivecompound. Preferably, ARL2 is detected in a biological fluid such ascell culture media and body fluids. This method may be applied toquantifying the level of ARL2 expression in a cell sample or individual.This information may be useful in determination of microtubule-relateddisorders, such as, but not limited to, cancer, human polycystic kidneydisease, Alzheimer disease, and Down syndrome.

An additional preferred aspect is a host cell recombinant forpolynucleotides capable of directing NBART expression. Preferably, thepolynucleotides capable of directing NBART expression are located in the5′ regulatory region of the NBART gene. Further preferably, thesepolynucleotides are located within 500 base pairs of the NBART codingregion. These polynucleotides preferably comprise a promoter sequence.

A further aspect of the present invention involves the isolation andpurification of NBART. An embodiment of the present invention relates tocompositions comprising NBART polypeptides. The method of producingNBART polypeptides comprises the steps of: i) transfecting a mammalianhost cell with a recombinant expression vector comprising apolynucleotide of the present invention, and ii) purifying the producedprotein. NBART may be isolated according to any technique known in theart or disclosed herein. Preferably, an antibody directed against NBARTor a fragment thereof, is bound to a chromatographic support to form anaffinity chromatography column.

Protein of SEQ ID NO:50 (Internal Designation Clone 644724181-21-1-0-A12-F) The cDNA of Clone 644724_(—)181-21-1-0-A12-F (SEQ IDNO:49) encodes NBTG protein of SEQ ID NO:50, comprising the amino acidsequence: MHPFYTRAATMIGEIAAAVSFISKFLRTKGLTSERQLQTFSQSLQELLAEHYKHHWFPEKPCKGSGYRCIRINHKMDPLIGQAAQRIGLSSQELFRLLPSELTLWVDPYEVSYRIGEDGSICVLYEASPAGGSTQNSTNVQMVDSRISCKEELLLGRTSPSKNYNMMTVSS. Accordingly, it will beappreciated that all characteristics and uses of the polypeptides of SEQID NO:50 described throughout the present application also pertain tothe polypeptides encoded by the human cDNA included in Clone644724_(—)181-21-1-0-A12-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:49described throughout the present application also pertain to the nucleicacids comprising the human cDNA in Clone 644724_(—)181-21-1-0-A12-F. Apreferred embodiment of the invention is directed toward thecompositions of SEQ ID NO:49, SEQ ID NO:50 and Clone644724_(—)181-21-1-0-A12-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

NBTG is a novel polymorphism variant of B-cell translocation gene 1protein (accession numbers P31607). The protein of the inventiondisplays an anti-proliferative domain:IGEIAAAVSFISKFLRTKGLTSERQLQTFSQSLQELLAEHYKHHWFPEKPCKGSGYRCIRINHKMDPLIGQAAQRIGLSSQELFRLLPSELTLWVDPYEVSYRIGEDGSICVLYEASPAGGSTQNSTNVQMVDSRISCKEELLLGRTSPSKNYNMMTVSS (SEQ ID NO:107). Accordingly, someembodiments of the present invention relate to polypeptides comprisingthe anti-proliferative domain.

The ability to negatively regulate cell proliferation is a necessity forall living organisms. Unicellular organisms must limit their replicationto the time when adequate nutrients and other environmental factors arepresent, and multicellular organisms must accurately shape and maintainthe architecture of their component tissues. The failure in amulticellular organism to provide adequate negative growth control inthe developmental period may result in a malformation, which may belethal. In the postdevelopmental period, such a failure may result inneoplasia. Because negative control is so critical, specific genes haveevolved whose role is actively antiproliferative.

NBTG is a member of a family of antiproliferative genes. NBTG is crucialto counteract the growth inducing elements and have the same importanceas proto-oncogenes in controlling cell division. Loss of NBTG isassociated with irregular cellular differentiation and proliferation orwith alteration of embryonic development. NBTG downregulates N1H3T3 cellproliferation when over expressed. NBTG gene is involved in achromosomal translocation in B-cell chronic lymphocytic leukemia. NBTGis expressed in tissues (lymphoid, liver, plasma) containingnon-dividing cells likely to re-enter the cell cycle upon stimuli,however, NBTG is barely detectable in fully differentiated tissues suchas brain and muscle.

An embodiment of the invention is directed to a composition comprising aNBTG polypeptide sequence of SEQ ID NO:50.

A further embodiment of the invention is directed to a compositioncomprising a NBTG polypeptide fragment having a biological activity ofinhibiting cell proliferation.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:49 encoding a NBTGpolypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a NBTG polypeptidefragment having a biological activity of inhibiting cell proliferation.

A further embodiment of the invention is directed to a compositioncomprising an antibody directed against the NBTG polypeptide or a NBTGpolypeptide fragment having biological activity. Preferably, theantibody recognizes a non-linear epitopes, and specifically binds to theC-terminal sequence of NBTG.

A preferred aspect of the invention is a host cell recombinant forpolynucleotides encoding a NBTG polypeptide or a biologically activefragment thereof. An additional preferred aspect is a host cellrecombinant for polynucleotides capable of directing NBTG expression.Preferably, the polynucleotides capable of directing NBTG expression arelocated in the 5′ regulatory region of the NBTG gene. Furtherpreferably, these polynucleotides are located within 500 base pairs ofthe NBTG coding region. These polynucleotides preferably comprise apromoter sequence. Techniques known in the art for introducingpolynucleotide sequences to endogenous sequences are described in U.S.Pat. No. 5,641,670 and PCT WO9629411, which disclosures are herebyincorporated by reference in their entireties. NBTG protein produced bysaid host cell may be used for in vitro detection and purificationmethods as well as diagnosis and in vivo applications.

An embodiment of the present invention relates to compositionscomprising NBTG polypeptides. The method of producing NBTG polypeptidescomprises the steps of: i) transfecting a mammalian host cell with arecombinant expression vector comprising a polynucleotide of the presentinvention, and ii) purifying the produced protein. The purification ofthe protein can be done following any technique well known to thoseskilled in the art. Preferably, an antibody directed against NBTG orpart thereof, more preferably, an antibody directed against theC-terminal sequence of NBTG polypeptide, may be bound to achromatographic support to form an affinity chromatography column.

In another embodiment, the invention provides methods and compositionsfor detecting the level of expression of NBTG mRNA. Quantification ofmRNA levels of NBTG polypeptides is useful for the diagnosis orprognosis of diseases associated with an altered expression of theprotein of the invention. Conditions, diseases or disorders associatedwith altered expression include, but are not limited to, aberrantcellular proliferation, such as cancer, psoriasis, blood vesselproliferative disorders, fibrotic disorders, and actinic lesions. Assaysfor the detection and quantification of the mRNA of the protein of theinvention are well known in the art (see, for example, Maniatis, Fitschand Sambrook, Molecular Cloning; A Laboratory Manual (1982), or CurrentProtocols in Molecular Biology, Ausubel, F. M. et al. (Eds), Wiley &Sons, Inc.). For example, the nucleic acid molecule or probe may belabeled by standard methods and added to a biological sample from apatient under conditions of formation of hybridization complexes. Afteran incubation period, the sample is washed and the amount of label (orsignal) associated with hybridization is quantified and compared with astandard value. If the amount of label in the patient sample issignificantly altered in comparison to the standard value, then thepresence of the associated condition, disease or disorder is indicated.Such assays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies and in clinical trialand to monitor the treatment of an individual patient. Once the presenceof a condition is established and a treatment protocol is initiated,diagnostic assays may be repeated on a regular basis to determine if thelevel of expression in the patient begins to approximate that which isobserved in a normal subject. The results obtained from successiveassays may be used to show the efficacy of treatment over a periodranging from several days to months.

In another embodiment, the invention relates to methods and compositionsfor detecting and quantifying the level of NBTG present in a particularbiological sample. These methods are useful for the diagnosis orprognosis of diseases associated with altered levels of the protein ofthe invention. Diagnostic assays to detect NBTG may require cells from abiopsy, in situ assay of cells from organ or tissue sections, or anaspirate of cells from a tumor or normal tissue. In addition, assays maybe conducted upon cellular extracts from organs, tissues, cells, urine,or serum or blood or any other body fluid or extract. Detection andquantification of NBTG polypeptides using either specific polyclonal ormonoclonal antibodies are known in the art. Examples of such techniquesinclude enzyme-linked immunoabsorbent assays (ELISAs), radioimmunoassays(RIAs), and fluorescence activated cell sorting (FACS).

NBTG has an antiproliferative ability, and thus may be employed to treatdiseases or pathological conditions associated with aberrant cellularproliferation and malignant conditions. The polypeptides may be employedto inhibit tumor growth and cell proliferation. NBTG may also beemployed to prevent scar formation at the site of wound healing.Restenosis, or re-occlusion of arterial walls after balloon angioplasty,may also be treated with NBTG as arteries re occlude through cellproliferation. Similarly angiogenesis into a tumor may be inhibited.

In one embodiment, NBTG or a biologically active fragment thereof isadministered to treat or prevent a condition associated with alteredNBTG expression or activity. Examples of such conditions include, butare not limited to, those described above. Most preferably, acomposition comprising a NBTG polypeptide is administered to anindividual suffering from abnormal or undesirable cell proliferation,such as, e.g., tumor growth, endothelial cell proliferation, andangiogenesis related to tumor growth. Tumors that can be treated withthe compositions of the present invention include, but are not limitedto, gliomas, amelanotic melanomas, prostate tumors and lung tumors.

In another embodiment, a pharmaceutical composition comprising asubstantially purified NBTG polypeptide in conjunction with apharmaceutical carrier may be administered to a subject to treat orprevent a condition associated with altered expression or activity ofthe endogenous protein including, but not limited to, those providedabove.

In another embodiment, a substance which modulates the activity of NBTGmay be administered to a subject to treat or prevent a conditionassociated with altered lifespan, expression, or activity of NBTGpolypeptides including, but not limited to, those listed above. In oneaspect, an antibody which specifically binds NBTG may be used as atargeting and delivery mechanism for bringing a pharmaceutical agent tocells or tissue which express NBTG like, but not limited to, the livercells.

Another embodiment of the subject invention provides compositions andmethods of selectively increasing the activity of the protein of theinvention. Activation of NBTG allows for the successful treatment and/ormanagement of diseases or biochemical abnormalities associated with NBTGactivity. Agonists, able to increase the expression or the activity ofthe protein of the invention, are useful in the treatment of diseasesassociated with cell proliferation, like, but not limited to, cancer,psoriasis, blood vessel proliferative disorders, fibrotic disorders, andactinic lesions. Alternatively, antagonist, able to decrease theexpression or the activity of NBTG, are useful in the treatment ofconditions characterized by insufficient cellular proliferation. Theconditions to be treated include, for example, osteoporosis, fragileskin and poor wound healing.

In an additional embodiment, a vector capable of expressing NBTG or apreferred fragment may be administered to a subject to treat or preventa condition associated with altered lifespan, expression, or activity ofthe protein of the invention including but not limited to, those listedabove. NBTG gene and gene products may also be employed for modulationof cellular growth. Due to their anti-proliferative effect they could beselectively administered or possibly inhibited when it is desirable tohave certain cells proliferate. An example would be a disorder relatedto the underproduction of certain cells, where proliferation anddifferentiation of theses cells would helps to treat the disorderslisted above.

In some embodiments, the invention also concerns a diagnostic kit fordetecting in vitro the presence of NBTG polypeptide. This kit comprises:a polyclonal or monoclonal antibody or fragment thereof thatspecifically binds a NBTG polypeptide; and optionally, ii) a reagentallowing the detection of the antigen-antibody complexes formed.Preferably, the antibody, or antibody fragment is detectably labeled.Such labels include fluorescent luminescent, and radioactive compounds,as well as enzymatic substrates. The optional reagent may provide adetectable signal in either bind to the antibody or react with the labelon the antibody. NBTG antibodies may be used to diagnose lowproliferative diseases such as osteoporosis, fragile skin and poor woundhealing. To diagnose such disorders, an appropriate biological samplecan be tested to determine the level of NBTG being produced.

In another embodiment, the current invention provides a method ofeffectively blocking proliferation or inhibiting the growth of a cell invivo or in vitro. Preferably, the present invention can be used to stopunrestrained cell proliferation and to eliminate as many tumor cells aspossible. The method will be performed by administering an antisenseoligonucleotide directed against NBTG to a cell. One strategy fordelivering antisense oligonucleotides to targeted cells involvesencapsulation or incorporation of the therapeutic bioactive molecules inliposomes, such as cationic liposomes. These liposomes are known toprovide a shield against nucleotide degradation in vivo and can betargeted to specific areas of the body at which point they slowlyrelease their contents. Alternatively, a polynucleotide constructcomprising plasmid DNA operably linked to the antisense oligonucleotidecould be used. The nucleotide sequences are administered in vivo in asuitable buffer or carrier solution known to those skilled in the art.

Another embodiment of the invention relates to composition and methodsusing polynucleotide sequences encoding the protein of the invention ora fragment thereof to establish transgenic model animals (D.melanogaster, M. musculus), by any method familiar to those skilled inthe art. By modulating in vivo the expression of the transgene withdrugs or modifier genes (activator or suppressor genes), animal modelscan be developed that mimic human hormone-dependent disorders such ascancers. These animal models would thus allow the identification ofpotential therapeutic agents for treatment of the disorders. Inaddition, recombinant cell lines derived from these transgenic animalsmay be used for similar approaches ex vivo.

Protein of SEQ ID NO:52 (Internal designation 583702 181-8-4-0-C8-F) ThecDNA of 583702_(—)181-8-4-0-C8-F (SEQ ID NO:51) encodes protein vITH1 ofSEQ ID NO:52, comprising the amino acid sequence:MPLPLPSAFVLSALQPSPTHSSSNTQRLPDRVTGGFSVNGQLIGNKARSPGQHDGTYFGRLGIANPATDFQLEVTPQNITLNPGFGGPVFSWRDQAVLRQDGVVVTINKKRNLVVSVDDGGTFEVVLHRVWKGSSVHQDFLGFYVLDSHRMSARTHGLLCIQFFHPIGFEVSDIHPGSDPTKPDATMVVRNRRLTVTRGLQKDYSKDPWHGAEVSCWFIHNNGAGLIDGAYTDYIVPDIF. Accordingly, itwill be appreciated that all characteristics and uses of polypeptides ofSEQ ID NO:52 described throughout the present application also pertainto the polypeptides encoded by the human cDNA included in Clone583702_(—)181-8-4-0-C8-F. In addition, it will be appreciated that allcharacteristics and uses of the polynucleotides of SEQ ID NO:51described throughout the present application also pertain to the nucleicacids comprising the human cDNA in Clone 583702_(—)181-8-4-0-C8-F. Apreferred embodiment of the invention is directed toward thecompositions of SEQ ID NO:51, SEQ ID NO:52, and583702_(—)181-8-4-0-C8-F. Also preferred are polypeptide fragmentshaving a biological activity as described herein and the polynucleotidesencoding the fragments.

The cDNA of SEQ ID NO:51 is a splice variant of the heavy chain 1 of thehuman inter-alpha-trypsin inhibitor (ITH1), encoding a 239 amino-acidprotein of SEQ ID NO:52 named vITH1.

Inter-alpha-trypsin inhibitor belongs to the superfamily of Kunitz-typeserine protease inhibitors in mammals. It is a glycosylated proteaseinhibitor with a polypeptide chain structure composed of two heavychains (HC1 and HC2) covalently linked to a light chain (bikunin).Bikunin functions as the protease inhibitor. vITH1 polypeptide is anovel splice variant of the heavy chain 1 that lacks the bikunin linkagesite. However, ITH1 binds hyaluronic acid (HA) protein. The connectivetissues are the main mammalian source of HA but it is also found in theextracellular matrix, cartilage, bone marrow, synovial fluid. Afterendocytosis, HA is normally catabolized by hyaluronidase digestion inthe liver.

An embodiment of the invention is directed to a composition comprising avITH1 polypeptide sequence of SEQ ID NO:52.

A further embodiment of the invention is directed to a compositioncomprising a vITH1 polypeptide fragment having a biological HA-bindingprotein activity.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence of SEQ ID NO:51 encoding a vITH1polypeptide.

A further embodiment of the invention is directed to a compositioncomprising a polynucleotide sequence encoding a vITH1 polypeptidefragment having a biological HA-binding protein activity.

A further embodiment of the invention is directed to a compositioncomprising an antibody directed against a vITH1 polypeptide sequence ofSEQ ID NO: 52 or a vITH1 polypeptide fragment having a biologicalHA-binding protein activity. Preferably, the antibody specifically bindsto vITH1 but not to ITH1. Preferably, the antibody specificallyrecognizes an epitope comprising the amino acids: VTGG (SEQ ID NO:108),TGGF (SEQ ID NO:109), GGFS (SEQ ID NO:110), VTGGFS (SEQ ID NO:111), MPLP(SEQ ID NO:112), PLPL (SEQ ID NO:113), PLPS (SEQ ID NO:114), LPSA (SEQID NO:115), or MPLPLPSA (SEQ ID NO:116).

An embodiment of the present invention relates to compositionscomprising vITH1 polypeptides. The method of producing vITH1polypeptides comprises the steps of: i) transfecting a mammalian hostcell with a recombinant expression vector comprising a polynucleotide ofthe present invention, and ii) purifying the produced protein. Thepurification of the protein can be done following any technique wellknown to those skilled in the art. Preferably, an antibody directedagainst vITH1 or fragment thereof, as described above, may be bound to achromatographic support to form an affinity chromatography column.

In another embodiment, the invention is directed to a method ofdetecting vITH1 polypeptide in a biological sample, said methodcomprising the steps of: i) contacting a biological sample with anantibody or antibody fragment that specifically binds vITH1 polypeptide;and ii) detecting the antigen-antibody complex formed. The antibody orantibody fragment may be monoclonal or polyclonal. In addition, theantibody or antibody fragment may be primarily or secondarily labeled bya detectable compound (e.g., radioactive, fluorescent, luminescent, orenzymatic) common in the art.

In a further embodiment, vITH1 polypeptide is used to purify HA. In suchmethod, vITH1 polypeptide is preferably covalently or non-covalentlyattached to a solid matrix and allowed to bind HA using techniques wellknown in the art. This method comprises the steps of: i) washing thesolid matrix to get rid of contaminants, ii) eluting the particle ofinterest using more stringent conditions. Additional aspects of thisembodiment include methods of using vITH1 polypeptide to detect andquantify HA using techniques common in the art. This method comprisesthe steps of: i) obtaining a biological sample suspected of containingHA, ii) contacting said sample with a vITH1 polypeptide or fragmentthereof under conditions suitable for binding of vITH1 and detecting thepresence or absence of HA by detecting vITH1. Preferably, vITH1polypeptide or fragment thereof is covalently attached to a detectablecompound. Alternatively, a detectable vITH1-specific antibody orfragment thereof may be used to detect vITH1. This embodiment is useful,for example, as a diagnostic tool for detecting the presence of HA forindividuals at risk of or suffering from a wide range of diseasesassociated with an abnormal HA storage. For example, it can be appliedin the diagnosis of conditions such as, but not limited to,hyaluronidase deficiency also known as type IX mucopolysaccharidosis,disorders of HA metabolism associated with generalized folding andthickening of the skin as described by Ramsden C A et al, J Pediatr 2000January; 136(1):62-68), liver diseases such as cirrhosis, renal failureand several lung diseases, arthritis diseases such as rheumatoidarthritis and osteoarthritis, and certain cancers such as mesotheliomaand Wilms' tumors. Such method is also useful to evaluate the severitydegree of such diseases. This kit comprises: a polyclonal or monoclonalantibody or fragment thereof that specifically binds a vITH1polypeptide; and optionally, ii) a reagent allowing the detection of theantigen-antibody complexes formed. Preferably, the antibody or antibodyfragment is detectably labeled. Such labels include fluorescent,luminescent, and radioactive compounds, as well as enzymatic substrates.The optional reagent may provide a detectable signal and either bind tothe antibody or react with the label on such antibody. To determine thelevel of HA and diagnose or manage such disorders, preferred appropriatebiological samples are serum, urine and synovial fluid in the case ofarthritis disorders.

An embodiment of the invention provides for a method of screening testsubstances for modulators of vITH1 expression. This method comprises thesteps of: i) contacting a cell with a test substance, and ii) comparingvITH1 expression in the cell after exposure to the test substance tothat of an unexposed control cell. vITH1 expression is determined bymethods common to the art or included herein, by detecting vITH1polynucleotides or polypeptides. An example of this method comprises thesteps of: i) culturing two equivalent cell samples, ii) adding a testsubstance to one of the cultures and not the other, iii) harvesting bothcultures at a specified time, iv) purifying the mRNA from each sample ofcells, v) comparing the level of vITH1 mRNA in each sample by Northernblot, RTPCR, or another method common to the art. The invention providesfor design and use of specific polynucleotide probes and primers, asdiscussed herein. An additional example comprises the steps of: i)having two equivalent cultures of cells, ii) adding a test substance toone of the cultures and not the other, iii) harvesting both cultures,iv) purifying the protein from each sample of cells, v) comparing thelevel of vITH1 polypeptides in each sample by Western blot,immunohistochemistry, or another method common to the art. The inventionprovides for design and use of specific antibodies and antibodyfragments, as discussed herein.

In another embodiment, a vITH1 polypeptide or a fragment thereof, may beused to screen for compounds that activate or inhibit vITH1 activity.This method comprises the steps of: i) contacting a vITH1 polypeptide orfragment thereof with a test substance and ii) monitoring vITH1activity. vITH1 binds to HA. Thus, vITH1 activity may be monitored uponaddition of the test substance by competitive binding assays with HA. Inthis aspect of the invention, a vITH1 polypeptide or fragment thereofmay be free in solution, affixed to a solid support, recombinantlyexpressed on or chemically attached to a cell surface, or locatedintracellularly. The formation of binding complexes between vITH1polypeptide and the compound being tested, may be measured by methodswell known to those skilled in the art, such as the BIAcore (Upsala,Sweden). Another technique provides for high throughput screening ofcompounds having suitable binding affinity to the protein of theinvention. Test substances that decrease vITH1 expression or activityare defined as inhibitors or antagonists of vITH1. Test substances thatincrease vITH1 expression or activity are defined as activators oragonists. Agents which modulate the expression or activity of the vITH1of the subject invention include, but are not limited to antisenseoligonucleotides, ribozymes, and antibodies. These agents may be madeand used according to methods well known in the art.

In another embodiment of the invention, the vITH1 polypeptide, fragmentsthereof, or vITH1 agonists are used to bind HA in vivo and remove thismolecule from the bloodstream. This method can be used to prevent ortreat disorders associated with abnormal storage of HA, including thosedue to a decrease hepatic clearance, an excessive synthesis or anincreased hyaluronidase activity, For example, vITH1 polypeptides,fragments thereof, or vITH1 agonists can be used in a compositiondelivered to an individual at risk of or suffering from, but not limitedto, hyaluronidase deficiency also known as type IXmucopolysaccharidosis, disorders of HA metabolism associated withgeneralized folding and thickening of the skin as described by Ramsden CA et al, J Pediatr 2000 January; 136(1):62-68), liver diseases such ascirrhosis, renal failure and several lung diseases. In this embodiment,such compositions can also be used in any other conditions associatedwith elevated serum levels of HA such as arthritis diseases such asrheumatoid arthritis and osteoarthritis, and certain cancers such as,but not limited to, mesothelioma and Wilms' tumors. This methodcomprises the step of: introducing an effective amount of vITH1polypeptide, fragment thereof or vITH1 agonist to the bloodstream of anindividual. In this embodiment, the vITH1 polypeptide may further beexpressed as a fusion protein with a polypeptide signal specifyingexcretion from the body. A preferred method of delivering vITH1polypeptides or biologically active fragments thereof to an individualincludes direct, intravenous injection of said polypeptides or fragmentsin a physiologically acceptable solution (e.g., pH-buffered isotonicsaline solutions, pH-buffered isotonic saline solutions modified byaddition of viscous elements such as glycerol). Alternatively, vITH1polynucleotides may be introduced to express vITH1 polypeptides in thebloodstream. This method comprises the steps of: i) constructing arecombinant viral vector corresponding to a portion of the genome of anadenovirus capable of infecting a cell operatively linked to thenucleotide sequence of the invention and a regulatory sequence directingits expression; ii) delivery of an effective amount of the recombinantadenoviral vector to an individual with or at risk of suffering diseasesor conditions mentioned above.

Another embodiment of the invention relates to animal models generatedby modulating the expression or activity of the present protein in oneor more tissues of the animal. These animals can be generated with anymethod of targeting overexpression or inactivation of vITH1, or bymodulating the in vivo expression of the transgene with drugs ormodifier genes (activator or suppressor genes). Such animals are usefulfor a number of purposes, because they represent an in vivo assay methodfor testing therapeutic candidate molecules potentially useful for thetreatment of various pathophysiological aspects of diseases specificallyrelated to the activity of vITH1. In addition, recombinant cell linesderived from these transgenic animals may be used for similar approachesex vivo.

Nested N-Terminal Fragments of Related Proteins Acrp30R1L and Acrp30R1

The cDNA of Accession No. AAB30232 comprising the nucleotide sequence:(SEQ ID NO: 117) ggaaaactatgcctggggccgacgctctgcccggctgctgccgctgaggaaagccgggacgcggagccccgccgagagcttctttgctccggacgcccctggacgtggcgggcagccgcgagggtaaccaccatgatcccctgggtgctcctggcctgtgccctcccctgtgctgctgacccactgcttggcgcctttgctcgcagggacttccggaaaggctcccctcaactggtctgcagcctgcctggcccccagggcccacccggccccccaggagccccagggccctcaggaatgatgggacgaatgggctttcctggcaaagacggccaagatggacacgacggcgaccggggggacagcggagaggaaggtccacctggccggacaggtaaccggggaaagccaggaccaaagggcaaagccggggccattgggcgggctggcccccgtggccccaagggggtcaacggtacccccgggaagcatggcacaccaggcaagaaggggcccaagggcaagaaaggggagccaggcctcccaggcccctgcagctgtggcagtggccataccaagtcagctttctcggtggcagtgaccaagagctacccacgggagcggctgcccatcaagtttgacaagattctgatgaacgagggtggccactacaatgcttccagcggcaagttcgtctgcggcgtgcctgggatctactacttcacctacgacatcacgctggccaacaagcacctggccatcggcctggtgcacaacggccagtaccgcatccggacctttgatgccaacaccggcaaccacgatgtggcctcaggctccaccatcctggctctcaagcagggtgacgaagtttggctgcagatcttctactcagagcagaacgggctcttctatgacccttactggacagacagcctctttacgggcttcctaatctatgccgaccaggatgaccccaacgaggtatagacatgccacggcggtcctccaggcagggaacaagcttctggacttgggcttacagagcaagaccccacaactgtaggctgggggtggggggtcgagtgagcggttctagcctcaggctcacctcctccgcctctttttttccccttcattaaatccaaac ctttttattca

encodes the protein Acrp30R1L comprising the amino acid sequence: (SEQID NO: 118) MIPWVLLACALPCAADPLLGAFARRDFRKGSPQLVCSLPGPQGPPGPPGAPGPSGMMGRMGFPGKDGQDGHDGDRGDSGEEGPPGRTGNRGKPGPKGKAGAIGRAGPRGPKGVNGTPGKHGTPGKKGPKGKKGEPGLPGPCSCGSGHTKSAFSVAVTKSYPRERLPIKFDKILMNEGGHYNASSGKFVCGVPGLYYFTYDITLANKHLALGLVHNGQYRIRTFDANTGNHDVASGSTILALKQGDEVWLQIFYSEQNGLFYDPYWTDSLFTGFLIYADQDDPNEV.Accordingly, it will be appreciated that all characteristics and uses ofpolypeptides of the aforesaid Acrp30R1L amino acid sequence describedthroughout the present application also pertain to the polypeptidesencoded by the nucleic acids included in the aforesaid Acrp30R1Lnucleotide sequence. Also preferred are fragments having a biologicalactivity as described therein and the polynucleotides encoding thefragments.Acrp30R1L is comprised of at least three regions including:1. an N-terminal putative signal sequence about from amino acids 1-20;2. a unique region about from amino acids 21-39;3. a collagen-like region about from amino acids 40-141; and4. a globular region of C1q homology about from amino acids 149-285.Acrp30R1L is comprised of several notable putative sites for proteolyticcleavage including:1. collagenase matrix metalloproteinase-1 (MMP-1) between amino acids46-47;2. plasmin between amino acids 125-126, 126-127, 131-132, and 132-133;and3. precerebellin processing protease between amino acids 133-134.

The cDNA of Accession No. AAZ45688 comprising the nucleotide sequence(SEQ ID NO: 119) gaattcggcacgagggccgcgagggtaaccaccatgatcccctgggtgctcctggcctgtgccctcccctgtgctgctgacccactgcttggcgcctttgctcgcagggacttccggaaaggctcccctcaactggtctgcagcctgcctggcccccagggcccacccggccccccaggagccccagggccctcaggaatgatgggacgaatgggctttcctggcaaagacggccaagatggacacgacggcgaccggggggacagcggagaggaaggtccacctggccggacagtgaccaagagctacccacgggagcggctgcccatcaagtttgacaagattctgatgaacgagggtggccactacaatgcttccagcggcaagttcgtctgcggcgtgcctgggatctactacttcacctacgacatcacgctggccaacaagcacctggccatcggcctggtgcacaacggccagtaccgcatccggacctttgatgccaacaccggcaaccacgatgtggcctcaggctccaccatcctggctctcaagcagggtgacgaagtttggctgcagatcttctactcagagcagaacgggctcttctatgacccttactggacagacagcctctttacgggcttcctaatctatgccgaccaggatgaccccaacgaggtatagacatgccacggcggtcctccaggcagggaacaagcttctggacttgggcttacagagcaagaccccacaactgtaggctgggggtggggggtcgagtgagcggttctagcctcaggctcacctcctctgcctctttttttccccttcattaaatccaaacctttttattcaaaaaaaaaaaaaaaaaaagatgcggccg

encodes the protein Acrp30R1 comprising the amino acid sequence (SEQ IDNO: 120) MIPWVLLACALPCAADPLLGAFARRDFRKGSPQLVCSLPGPQGPPGPPGAPGPSGMMGRMGFPGKDGQDGHDGDRGDSGEEGPPGRTVTKSYPRERLPIKFDKILMNEGGHYNASSGKFVCGVPGIYYFTYDITLANKHLAIGLVHNGQYRIRTFDANTGNHDVASGSTILALKQGDEVWLQIFYSEQNGLFYDPYWTDS LFTGFLIYADQDDPNEV.

Accordingly, it will be appreciated that all characteristics and uses ofpolypeptides of the aforesaid Acrp30R1 amino acid sequence describedthroughout the present application also pertain to the polypeptidesencoded by the nucleic acids included in the aforesaid Acrp30R1nucleotide sequence. Also preferred are fragments having a biologicalactivity as described therein and the polynucleotides encoding thefragments.

Acrp30R1 is comprised of at least three regions including:

1. an N-terminal putative signal sequence about from amino acids 1-20;

2. a unique region about from amino acids 21-39;

3. a collagen-like region about from amino acids 40-87; and

4. a globular region of C1q homology about from amino acids 88-217.

Acrp30R1 retains the putative site for MMP-1 cleavage present inAcrp30R1L but lacks the putative sites in Acrp30R1L for cleavage byplasmin and precerebellin processing protease.

Acrp30R1L and Acrp30R1 are related proteins resulting from differentialsplicing of a single gene. Acrp30R1L and Acrp30R1 are comprised of acollagen domain and a C1q homology domain. Acrp30R1 lacks an internal 68amino acid segment present in Acrp30R1L. The N-terminal fragments ofAcrp30R1L and Acrp30R1 generated in vivo by proteolytic cleavage of thefull-length protein have unexpected and novel function as describedbelow.

Chemokines are chemotactic cytokines that signal through Gprotein-coupled receptors (GPCR). Secondary lymphoid-tissue chemokine(SLC) is a polypeptide of 134 amino acids including a putative signalsequence of 23 amino acids (Nagira, M et al., J Biol Chem 272:19518-24(1997) which disclosure is hereby incorporated by reference in itsentirety). SLC is highly expressed by high endothelial venules,specialized vessels involved in the homing of lymphocytes from the bloodinto lymph nodes and Peyer's patches (Murphy, P M et al.,Pharmacological Reviews 52:145-176 (2000); Dieu-Nosjean, M C et al., JLeukoc Biol 66:252-62 (1999) which disclosures are hereby incorporatedby reference in their entirety). SLC is chemotactic for T cells, Bcells, and mature dendritic cells. SLC functions as an importantmediator of physiological lymphocyte recirculation in vivo (Gunn, M D etal, Proc Natl Acad Sci USA 95:258-63 (1998); Gunn, M D et al., J Exp Med189:451-60 (1999) which disclosures are hereby incorporated by referencein their entirety). Recently it was shown that neurodegenerative stressinduces neuronal expression of SLC and that SLC acts on microglia(Biber, K et al., Glia 34:121-33 (2001) which disclosure is herebyincorporated by reference in its entirety).

SLC binds to chemokine receptor CCR7 (Yoshida, R et al., J Biol Chem273:7118-22 (1998) which disclosure is hereby incorporated by referencein its entirety). EBI1-ligand chemokine (ELC) also binds to CCR7.Recently it was shown that SLC also binds to the recently identifiedchemokine receptor CCR10 (Gosling, J et al., J Immunol 164:2851-6 (2000)which disclosure is hereby incorporated by reference in its entirety).SLC and ELC bind competitively and only to CCR7 and CCR10.

SLC has been implicated in chronic inflammation, including rheumatoidarthritis (Patel, D D et al., Clin Immunol 99:43-52 (2001) whichdisclosure is hereby incorporated by reference in its entirety) andprediabetic NOD mice (Hjelmstrom, P et al., Am J Pathol 156:1133-8(2000) which disclosure is hereby incorporated by reference in itsentirety). SLC (and ELC) enhance the replication of HIV-1 in secondarylymphoid tissues (Nagira, M et al., Virology 264:422-6 (1999) whichdisclosure is hereby incorporated by reference in its entirety). SLC hasbeen shown to suppress proliferation of myeloid progenitor cells (Kim, CH et al., J Leukoc Biol 66:455-61 (1999) which disclosure is herebyincorporated by reference in its entirety). ELC (but not SLC) has beenimplicated in human atheriosclerosis (Reape, T J et al., Am J Pathol154:365-74 (1999) which disclosure is hereby incorporated by referencein its entirety).

SLC and ELC have been found to facilitate anti-tumor responses in vivo.SLC was found to enhance the anti-tumor response in several mouse models(Kirk, C J et al., Cancer Res 61:2062-70 (2001); Nomura, T et al., Int JCancer 91:597-606 (2001); Sharma, S et al., J Immunol 164:4558-63 (2000)which disclosures are hereby incorporated by reference in theirentirety). ELC was found to mediate tumor rejection of murine breastcancer cells (Braun, S E et al., J Immunol 164:4025-31 (2000) whichdisclosure is hereby incorporated by reference in its entirety).

Acrp30R1L and Acrp30R1 are characterized by an amino acid motif that isotherwise specific for chemokine SLC, as determined by BLAST analysis ofthe public protein database. The specificity of this motif for SLCextends across species. The motif, encompassing amino acids 21 to 46 ofAcrp30R1L and Acrp30R1 (and therefore N-terminal to the putativeprotease cleavage sites within Acrp30R1L and Acrp30R1) is:RxxRKxxPxLxCSxP (SEQ ID NO:121) (where “x” is an unassigned amino acid).This specific motif is conserved between human (Accession Nos. AAY12316,O00585, AAG03773, AAW87589), mouse (O09006, AAG45834) and pig (AAW50886)SLC (encompassing amino acids 46 to 60 of SLC). This motif is inferredto be important for SLC function, namely for binding to chemokinereceptors CCR7 and CCR10 and, as a corollary, for its competitivebinding with chemokine ELC to said receptors. N-terminal polypeptidefragments of Acrp30R1L and Acrp30R1 comprising said motifnon-productively bind to CCR7 and CCR10 and in so doing antagonize SLCand ELC function through CCR7 and CCR10.

The instant invention is based on the discovery that N-terminalpolypeptide fragments of the full-length Acrp30R1L or Acrp30R1polypeptides comprising said motif have unexpected effects relating toantagonism of SLC and ELC chemokine function. These effects areunexpected and surprising given that full-length Acrp30R1L or Acrp30R1polypeptide typically manifests no or significantly reduced effects. Tothe extent that any effect is manifested by full-length Acrp30R1L orAcrp30R1 polypeptide, the levels of full-length polypeptide required foran effect render it unfeasible in most instances as a potentialtreatment for humans at this time. In contrast, N-terminal polypeptidefragments of full-length Acrp30R1L or Acrp30R1 polypeptides comprisingsaid motif are radically more effective and thus can be provided atlevels that are feasible for treatments in humans.

Thus, the invention is drawn to Acrp30R1L and Acrp30R1 polypeptidefragments, polynucleotides encoding said Acrp30R1L and Acrp30R1polypeptide fragments, vectors comprising said Acrp30R1L and Acrp30R1polynucleotides, and cells recombinant for said Acrp30R1L and Acrp30R1polynucleotides, as well as to pharmaceutical and physiologicallyacceptable compositions comprising said Acrp30R1L and Acrp30R1polypeptide fragments and methods of administering said Acrp30R1L andAcrp30R1 pharmaceutical and physiologically acceptable compositions inorder to reduce chronic inflammation, reduce atheriosclerosis, reduceHIV replication in secondary lymphoid tissue, increase myelopoiesis, ortreat SLC- or ELC-related diseases or disorders. Assays for identifyingantagonists of said SLC- or ELC-related activity are also part of theinvention.

In a first aspect, the invention features a purified, isolated, orrecombinant Acrp30R1L or Acrp30R1 polypeptide fragment that that hassignificantly greater activity than a full-length Acrp30R1L or Acrp30R1polypeptide, wherein said activity is selected from but not restrictedto antagonism of SLC function or antagonism of ELC function. Inpreferred embodiments, Acrp30R1L polypeptide fragments having unexpectedactivity are selected from amino acids 21-46, 21-47, 21-48, 21-49,21-50, 21-51, 21-52, 21-53, 21-54, 21-55, 21-56, 21-57, 21-58, 21-59,21-60, 21-61, 21-62, 21-63, 21-64, 21-65, 21-66, 21-67, 21-68, 21-69,21-70, 21-71, 21-72, 21-73, 21-74, 21-75, 21-76, 21-77, 21-78, 21-79,21-80, 21-81, 21-82, 21-83, 21-84, 21-85, 21-86, 21-87, 21-88, 21-89,21-90, 21-91, 21-92, 21-93, 21-94, 21-95, 21-96, 21-97, 21-98, 21-99,21-100, 21-101, 21-102, 21-103, 21-104, 21-105, 21-106, 21-107, 21-108,21-109, 21-110, 21-111, 21-112, 21-113, 21-114, 21-115, 21-116, 21-117,21-118, 21-119, 21-120, 21-121, 21-122, 21-123, 21-124, 21-125, 21-126,21-127, 21-128, 21-129, 21-130, 21-131, 21-132, or 21-133 of aforesaidAcrp30R1L amino acid sequence and wherein amino acid 21 is taken to beunderstood to denote the N-terminal amino acid of full-length matureAcrp30R1L polypeptide lacking the signal peptide.

In other preferred embodiments, Acrp30R1 polypeptide fragments havingunexpected activity are selected from amino acids 21-46, 21-47, 21-48,21-49, 21-50, 21-51, 21-52, 21-53, 21-54, 21-55, 21-56, 21-57, 21-58,21-59, 21-60, 21-61, 21-62, 21-63, 21-64, 21-65, 21-66, 21-67, 21-68,21-69, 21-70, 21-71, 21-72, 21-73, 21-74, 21-75, 21-76, 21-77, 21-78,21-79, 21-80, 21-81, 21-82, 21-83, 21-84, 21-85, 21-86, or 21-87 ofaforesaid Acrp30R1 amino acid sequence and wherein amino acid 21 istaken to be understood to denote the N-terminal amino acid offull-length mature Acrp30R1 polypeptide lacking the signal peptide.

In yet other preferred embodiments, Acrp30R1L polypeptide fragmentshaving unexpected activity are selected from amino acids 21-46, 21-125,21-126, 21-131, 21-132, and 21-133 of aforesaid Acrp30R1L amino acidsequence and wherein amino acid 21 is taken to be understood to denotethe N-terminal amino acid of full-length mature Acrp30R1L polypeptide.In still other preferred embodiments, Acrp30R1 polypeptide fragmentshaving unexpected activity are selected from amino acids 21-46 and 21-87of aforesaid Acrp30R1 amino acid sequence and wherein amino acid 21 istaken to be understood to denote the N-terminal amino acid offull-length mature Acrp30R1 polypeptide.

In other further preferred embodiments, said polypeptide fragmentcomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the correspondingconsecutive amino acids of aforesaid Acrp30R1L amino acid sequence oraforesaid Acrp30R1 amino acid sequence.

In other preferred embodiments, said Acrp30R1L polypeptide fragments aremade by recombinant means or by proteolytic cleavage of full-lengthmature Acrp30R1L polypeptide. In other preferred embodiments, saidAcrp30R1 polypeptide fragments are made by recombinant means or byproteolytic cleavage of full-length mature Acrp30R1 polypeptide lackingthe signal peptide.

Particularly preferred proteolytic fragment of full-length matureAcrp30R1L polypeptide lacking the signal peptide is about amino acids21-46 made by collagenase cleavage of aforesaid Acrp30R1L amino acidsequence at about position 46 and wherein amino acid 21 is taken to beunderstood to denote the N-terminal amino acid of full-length matureAcrp30R1L polypeptide lacking the signal peptide. Other particularlypreferred proteolytic fragment of full-length mature Acrp30R1Lpolypeptide lacking the signal peptide is about amino acids 21-46 madeby MMP-1 cleavage of aforesaid Acrp30R1L amino acid sequence at aboutposition 46 and wherein amino acid 21 is taken to be understood todenote the N-terminal amino acid of full-length mature Acrp30R1Lpolypeptide lacking the signal peptide. Other particularly preferredproteolytic fragment of full-length mature Acrp30R1L polypeptide lackingthe signal peptide is about amino acids 21-125 made by plasmin cleavageof aforesaid Acrp30R1L amino acid sequence at about position 125 andwherein amino acid 21 is taken to be understood to denote the N-terminalamino acid of full-length mature Acrp30R1L polypeptide lacking thesignal peptide. Other particularly preferred proteolytic fragment offull-length mature Acrp30R1L polypeptide lacking the signal peptide isabout amino acids 21-126 made by plasmin cleavage of aforesaid Acrp30R1Lamino acid sequence at about position 126 and wherein amino acid 21 istaken to be understood to denote the N-terminal amino acid offull-length mature Acrp30R1L polypeptide lacking the signal peptide.Other particularly preferred is proteolytic fragment of full-lengthmature Acrp30R1L polypeptide lacking the signal peptide is about aminoacids 21-131 made by plasmin cleavage of aforesaid Acrp30R1L amino acidsequence at about position 131. Other particularly preferred proteolyticfragment of full-length mature Acrp30R1L polypeptide lacking the signalpeptide is about amino acids 21-132 made by plasmin cleavage ofaforesaid Acrp30R1L amino acid sequence at about position 132 andwherein amino acid 21 is taken to be understood to denote the N-terminalamino acid of full-length mature Acrp30R1L polypeptide lacking thesignal peptide. Other particularly preferred proteolytic fragment offull-length mature Acrp30R1L polypeptide lacking the signal peptide isabout amino acids 21-133 made by precerebellin processing proteasecleavage of aforesaid Acrp30R1L amino acid sequence at about position133 and wherein amino acid 21 is taken to be understood to denote theN-terminal amino acid of full-length mature Acrp30R1L polypeptidelacking the signal peptide.

Particularly preferred proteolytic fragment of full-length matureAcrp30R1 polypeptide lacking the signal peptide is about amino acids21-46 made by collagenase cleavage of aforesaid Acrp30R1 amino acidsequence at about position 46 and wherein amino acid 21 is taken to beunderstood to denote the N-terminal amino acid of full-length matureAcrp30R1 polypeptide lacking the signal peptide. Other particularlypreferred proteolytic fragment of full-length mature Acrp30R1polypeptide lacking the signal peptide is about amino acids 21-46 madeby MMP-1 cleavage of aforesaid Acrp30R1 amino acid sequence at aboutposition 46 and wherein amino acid 21 is taken to be understood todenote the N-terminal amino acid of full-length mature Acrp30R1polypeptide lacking the signal peptide.

In a second aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of, said Acrp30R1L or Acrp30R1 polypeptidefragment described in the first aspect and, alternatively, apharmaceutical or physiologically acceptable diluent.

In a third aspect, the invention features a method of preventing ortreating an immune-related disorder comprising providing oradministering to an individual in need of such treatment saidpharmaceutical or physiologically acceptable composition described inthe second aspect. Preferably, said immune-related disorder is selectedfrom but not restricted to allograft rejection or delayed typehypersensitivity.

In a fourth aspect, the invention features a method of preventing ortreating atheriosclerosis comprising providing or administering to anindividual in need of such treatment said pharmaceutical orphysiologically acceptable composition described in the second aspect.

In a fifth aspect, the invention features a method of preventing ortreating the inflammation associated with ischemic neurodegeneration inbrain tissue comprising providing or administering to an individual inneed of such treatment said pharmaceutical or physiologically acceptablecomposition described in the second aspect.

In a sixth aspect, the invention features a method of preventing ortreating the inflammation associated with stroke comprising providing oradministering to an individual in need of such treatment saidpharmaceutical or physiologically acceptable composition described inthe second aspect.

In a seventh aspect, the invention features a method of preventing ortreating the inflammation associated with myocardial infarctioncomprising providing or administering to an individual in need of suchtreatment said pharmaceutical or physiologically acceptable compositiondescribed in the second aspect.

In an eighth aspect, the invention features a method of preventing ortreating the inflammation associated with reperfusion injury comprisingproviding or administering to an individual in need of such treatmentsaid pharmaceutical or physiologically acceptable composition describedin the second aspect.

In a ninth aspect, the invention features a method of preventing ortreating HIV infection comprising providing or administering to anindividual in need of such treatment said pharmaceutical orphysiologically acceptable composition described in the second aspect.

In a tenth aspect, the invention features a method of preventing ortreating an inflammation-related disorder comprising providing oradministering to an individual in need of such treatment saidpharmaceutical or physiologically acceptable composition described inthe second aspect. Preferably, said inflammation-related disorder isselected from but not restricted to rheumatoid arthritis, inflammatorybowel disease, insulin dependent diabetes mellitus (Type 1 diabetes),systemic lupus erythematosus, psoriasis, allergic asthma, or septicshock.

In an eleventh aspect, the invention features a method of facilitatingengraftment and expansion of infused hematopoietic stem cells or myeloidprogenitor cells comprising providing or administering to an individualin need of such treatment said pharmaceutical or physiologicallyacceptable composition described in the second aspect.

In a twelfth aspect, the invention features a method of facilitatingengraftment and expansion within NOD/scid mice of infused humanhematopoietic stem cells or myeloid progenitor cells related to thepreconditioning regimen comprising a xenogeneic in vivo model of humanlymphoid and myeloid leukemia (Dialynas, D P et al., Blood 97:3218-25(2001) which disclosure is hereby incorporated by reference in itsentirety).

In a thirteenth aspect, the invention features a method of using saidAcrp30R1L or Acrp30R1 polypeptide fragment described in the first aspectin in vitro cell migration assays as an internal control for intact CCR7or CCR10 function.

The invention further features a method of using said Acrp30R1L orAcrp30R1 polypeptide fragment described in the first aspect in in vitrocell migration assays as an internal control for an intact T cell, Bcell, or mature dendritic cell chemotactic response (Patel, D D et al.,Clin Immunol 99:43-52 (2001) which disclosure is hereby incorporated byreference in its entirety).

In a fourteenth aspect, the invention provides for an antibody thatspecifically binds said preferred polypeptide fragment of matureAcrp30R1L lacking the signal peptide but not to full-length matureAcrp30R1L polypeptide lacking the signal peptide. Further preferred issaid antibody that recognizes a non-conformational or conformationalepitope of said preferred polypeptide fragment of mature Acrp30R1Llacking the signal peptide. Further preferred is said antibody thatneutralizes the capacity of said preferred polypeptide fragment ofmature Acrp30R1L lacking the signal peptide to antagonize SLC or ELCchemokine function.

Further preferred is a method wherein a mouse is immunized with saidpreferred polypeptide fragment of mature Acrp30R1L lacking the signalpeptide. Further preferred is a method wherein monoclonal antibodiesfrom said mouse are screened for binding to said preferred polypeptidefragment of mature Acrp30R1L lacking the signal peptide but not tofull-length mature Acrp30R1L polypeptide lacking the signal peptide.Further preferred is a method wherein monoclonal antibodies derived fromsaid mouse are screened by enzyme-linked immunosorbent assay (ELISA) forbinding to said preferred polypeptide fragment of mature Acrp30R1L butnot to mature Acrp30R1L polypeptide lacking the signal peptide. Furtherpreferred is a method wherein said antibody is screened for the capacityto sterically or allosterically neutralize the capacity of saidpreferred polypeptide fragment of mature Acrp30R1L lacking the signalpeptide to antagonize SLC or ELC chemokine function. Further preferredis a method of humanizing said monoclonal antibody. Methods ofgenerating said monoclonal antibody and of establishing specificity bymethods including ELISA are well known to those skilled in the art.Methods of screening said antibody for neutralization of the capacity ofsaid preferred polypeptide fragment of mature Acrp30R1L lacking thesignal peptide to antagonize SLC or ELC chemokine function are wellknown to those skilled in the art and include, but are not limited to:contacting the antibody with said preferred polypeptide fragment ofmature Acrp30R1L lacking the signal peptide, incubation of saidcontacted Acrp30R1L polypeptide fragment with radiolabeled SLC or ELC inthe presence of CCR7 or CCR10 chemokine receptor, and determination ofwhether said antibody contact blocks the capacity of said preferredpolypeptide fragment of mature Acrp30R1L lacking the signal peptide tocompeted with SLC or ELC for binding to CCR7 or CCR10 (Gosling, J etal., J Immunol 164:2851-6 (2000) which disclosure is hereby incorporatedby reference in its entirety). Methods of humanizing said monoclonalantibody are well known to those skilled in the art.

In a fifteenth aspect, the invention provides for an antibody thatspecifically binds said preferred polypeptide fragment of Acrp30R1 butnot full-length mature Acrp30R1 polypeptide lacking the signal peptide.Further preferred is said antibody that recognizes a non-conformationalor conformational epitope of said preferred polypeptide fragment ofAcrp30R1. Further preferred is said antibody that neutralizes thecapacity of said preferred Acrp30R1 polypeptide fragment to antagonizeSLC or ELC chemokine function.

Further preferred is a method wherein a mouse is immunized with saidpreferred polypeptide fragment of mature Acrp30R1 lacking the signalpeptide. Further preferred is a method wherein monoclonal antibodiesfrom said mouse are screened for binding to said preferred polypeptidefragment of mature Acrp30R1 lacking the signal peptide but not tofull-length mature Acrp30R1 polypeptide lacking the signal peptide.Further preferred is a method wherein monoclonal antibodies derived fromsaid mouse are screened by enzyme-linked immunosorbent assay (ELISA) forbinding to said preferred polypeptide fragment of mature Acrp30R1 butnot to mature Acrp30R1 polypeptide lacking the signal peptide. Furtherpreferred is a method wherein said antibody is screened for the capacityto sterically or allosterically neutralize the capacity of saidpreferred polypeptide fragment of mature Acrp30R1 lacking the signalpeptide to antagonize SLC or ELC chemokine function. Further preferredis a method of humanizing said monoclonal antibody. Methods ofgenerating said monoclonal antibody and of establishing specificity bymethods including ELISA are well known to those skilled in the art.Methods of screening said antibody for neutralization of the capacity ofsaid preferred polypeptide fragment of mature Acrp30R1 lacking thesignal peptide to antagonize SLC or ELC chemokine function are wellknown to those skilled in the art and include, but are not limited to:contacting the antibody with said preferred polypeptide fragment ofmature Acrp30R1 lacking the signal peptide, incubation of said contactedAcrp30R1 polypeptide fragment with radiolabeled SLC or ELC in thepresence of CCR7 or CCR10 chemokine receptor, and determination ofwhether said antibody contact blocks the capacity of said preferredpolypeptide fragment of mature Acrp30R1 lacking the signal peptide tocompeted with SLC or ELC for binding to CCR7 or CCR10 (Gosling, J etal., J Immunol 164:2851-6 (2000) which disclosure is hereby incorporatedby reference in its entirety). Methods of humanizing said monoclonalantibody are well known to those skilled in the art.

In a sixteenth aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of, said antibody of the fourteenth aspectdirected to said preferred Acrp30R1L polypeptide fragment and,alternatively, a pharmaceutical or physiologically acceptable diluent.

In a seventeenth aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of, said antibody of the fifteenth aspectdirected to said preferred Acrp30R1 polypeptide fragment and,alternatively, a pharmaceutical or physiologically acceptable diluent.

In an eighteenth aspect, the invention features a method of using saidantibody specific for said preferred Acrp30R1L or Acrp30R1 polypeptidefragment described in the fourteenth or sixteenth aspect in a method ofmeasuring the amount of said Acrp30R1L or Acrp30R1 polypeptide fragmentin a clinical sample for diagnosis of immune dysfunction. Preferably,said clinical sample is selected from the group consisting of blood,serum, plasma, urine, and saliva. Preferably, said immune dysfunction isselected from but not restricted to recurrent infection, innateimmunodeficiency, or acquired immunodeficiency. Elevated levels of saidAcrp30R1L or Acrp30R1 polypeptide fragment are expected to becontributory to said immune dysfunction.

In a nineteenth aspect, the invention features a method of using saidantibody specific for said preferred Acrp30R1L or Acrp30R1 polypeptidefragment described in the fourteenth or sixteenth aspect in a method ofmeasuring the amount of said Acrp30R1L or Acrp30R1 polypeptide fragmentin a clinical sample for cancer stratification. Preferably, saidclinical sample is selected from but not restricted to blood, serum,plasma, urine, and saliva. Preferably, said cancer is selected from butnot restricted to melanoma, squamous cell carcinoma of the skin, breastcarcinoma, lung small-cell carcinoma, colon carcinoma, Hodgkin'slymphoma, non-Hodgkin's lymphoma, prostatic carcinoma, pancreaticcarcinoma, osteosarcoma, uterine carcinoma, ovarian carcinoma,chondrosarcoma, endometrial cancer, testicular carcinoma, renalcarcinoma, hepatic carcinoma, lung non-small-cell carcinoma, T cellacute lymphoblastic leukemia, B cell acute lymphoblastic leukemia, acutemyeloid leukemia, chronic lymphocytic leukemia, or multiple myeloma.Elevated levels of said Acrp30R1L or Acrp30R1 are expected to becontributory to an impaired anti-tumor response.

In a twentieth aspect, the invention features a method of treatingcancer characterized by diagnostically determined elevated level of saidpreferred Acrp30R1L or Acrp30R1 polypeptide fragment comprisingproviding or administering to an individual in need of such treatmentsaid pharmaceutical or physiologically acceptable composition describedin the sixteenth or seventeenth aspect wherein the antibody comprisingsaid composition neutralizes the capacity of said preferred Acrp30R1L orAcrp30R1 polypeptide fragment to antagonize SLC or ELC chemokinefunction. Said neutralization is expected to facilitate the in vivoanti-tumor response. Preferably, said cancer is selected from but notrestricted to melanoma, squamous cell carcinoma of the skin, breastcarcinoma, lung small-cell carcinoma, colon carcinoma, Hodgkin'slymphoma, non-Hodgkin's lymphoma, prostatic carcinoma, pancreaticcarcinoma, osteosarcoma, uterine carcinoma, ovarian carcinoma,chondrosarcoma, endometrial cancer, testicular carcinoma, renalcarcinoma, hepatic carcinoma, lung non-small-cell carcinoma, T cellacute lymphoblastic leukemia, B cell acute lymphoblastic leukemia, acutemyeloid leukemia, chronic lymphocytic leukemia, or multiple myeloma.

In a twenty-first aspect, the invention provides for a non-antibodycompound that specifically binds said preferred polypeptide fragment ofmature Acrp30R1L lacking the signal peptide but not to full-lengthmature Acrp30R1L polypeptide lacking the signal peptide. Preferred saidcompound is selected from but not restricted to small molecular weightorganic or inorganic compound, protein, peptide, carbohydrate, or lipid.Further preferred is said antibody that recognizes a non-conformationalor conformational epitope of said preferred polypeptide fragment ofmature Acrp30R1L lacking the signal peptide. Further preferred is saidantibody that neutralizes the capacity of said preferred polypeptidefragment of mature Acrp30R1L lacking the signal peptide to antagonizeSLC or ELC chemokine function.

Further preferred are methods of screening for one or more compoundscapable of neutralizing the capacity of said preferred polypeptidefragment of mature Acrp30R1L lacking the signal peptide to antagonizeSLC or ELC chemokine function. Said methods are well known to thoseskilled in the art and include, but are not limited to: contacting thecompound with said preferred polypeptide fragment of mature Acrp30R1Llacking the signal peptide, incubation of said contacted Acrp30R1Lpolypeptide fragment with radiolabeled SLC or ELC in the presence ofCCR7 or CCR10 chemokine receptor, and determination of whether saidcontact with compound blocks the capacity of said preferred polypeptidefragment of mature Acrp30R1L lacking the signal peptide to competed withSLC or ELC for binding to CCR7 or CCR10 (Gosling, J et al., J Immunol164:2851-6 (2000) which disclosure is hereby incorporated by referencein its entirety).

In a twenty-second aspect, the invention provides for a non-antibodycompound that specifically binds said preferred polypeptide fragment ofmature Acrp30R1 lacking the signal peptide but not to full-length matureAcrp30R1 polypeptide lacking the signal peptide. Preferred said compoundis selected from but not restricted to small molecular weight organic orinorganic compound, protein, peptide, carbohydrate, or lipid. Furtherpreferred is said antibody that recognizes a non-conformational orconformational epitope of said preferred polypeptide fragment of matureAcrp30R1 lacking the signal peptide. Further preferred is said antibodythat neutralizes the capacity of said preferred polypeptide fragment ofmature Acrp30R1 lacking the signal peptide to antagonize SLC or ELCchemokine function.

Further preferred are methods of screening for one or more compoundscapable of neutralizing the capacity of said preferred polypeptidefragment of mature Acrp30R1 lacking the signal peptide to antagonize SLCor ELC chemokine function. Said methods are well known to those skilledin the art and include, but are not limited to: contacting the compoundwith said preferred polypeptide fragment of mature Acrp30R1 lacking thesignal peptide, incubation of said contacted Acrp30R1L polypeptidefragment with radiolabeled SLC or ELC in the presence of CCR7 or CCR10chemokine receptor, and determination of whether said contact withcompound blocks the capacity of said preferred polypeptide fragment ofmature Acrp30R1L lacking the signal peptide to competed with SLC or ELCfor binding to CCR7 or CCR10 (Gosling, J et al., J Immunol 164:2851-6(2000) which disclosure is hereby incorporated by reference in itsentirety).

In a twenty-third aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of, said compound of the twenty-firstaspect directed to said preferred Acrp30R1L polypeptide fragment and,alternatively, a pharmaceutical or physiologically acceptable diluent.

In a twenty-fourth aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of, said antibody of the twenty-secondaspect directed to said preferred Acrp30R1 polypeptide fragment and,alternatively, a pharmaceutical or physiologically acceptable diluent.

In a twenty-fifth aspect, the invention features a method of treatingcancer characterized by diagnostically determined elevated level of saidpreferred Acrp30R1L or Acrp30R1 polypeptide fragment comprisingproviding or administering to an individual in need of such treatmentsaid pharmaceutical or physiologically acceptable composition describedin the twenty-third or twenty-fourth aspect wherein the compoundcomprising said composition neutralizes the capacity of said preferredAcrp30R1L or Acrp30R1 polypeptide fragment to antagonize SLC or ELCchemokine function. Said neutralization is expected to facilitate the invivo anti-tumor response. Preferably, said cancer is selected from butnot restricted to melanoma, squamous cell carcinoma of the skin, breastcarcinoma, lung small-cell carcinoma, colon carcinoma, Hodgkin'slymphoma, non-Hodgkin's lymphoma, prostatic carcinoma, pancreaticcarcinoma, osteosarcoma, uterine carcinoma, ovarian carcinoma,chondrosarcoma, endometrial cancer, testicular carcinoma, renalcarcinoma, hepatic carcinoma, lung non-small-cell carcinoma, T cellacute lymphoblastic leukemia, B cell acute lymphoblastic leukemia, acutemyeloid leukemia, chronic lymphocytic leukemia, or multiple myeloma.

CRPF Polypeptides CRPF-1, CRPF-2A, CRPF-2B, CRPF-2C, and CRPF-3

CRPF polypeptide CRPF-1 comprises the amino acid sequence: SHHH (SEQ IDNO:122). CRPF polypeptide CRPF-2A comprises the amino acid sequence:AANSKVAFSAVRSTNH (SEQ ID NO:123). CRPF polypeptide CRPF-2B comprises theamino acid sequence: AANSKVAFSAVR (SEQ ID NO:124). CRPF polypeptideCRPF-2C comprises the amino acid sequence: STNH (SEQ ID NO:125). CRPFpolypeptide CRPF-3 comprises the amino acid sequence: SGSAKVAFSATRSTNH(SEQ ID NO:126). Also preferred are polynucleotides encoding the CRPFpolypeptides.

The CRPF polypeptides are related to cerebellin. The CRPF polypeptideshave unexpected and novel function as described below.

Cerebellin is a 16 amino acid polypeptide liberated by specificproteolytic cleavage from a precursor polypeptide, precerebellin (Urade,Y et al., Proc Natl Acad Sci USA 88:1069-1073 (1991) which disclosure ishereby incorporated by reference in its entirety). Cerebellin isconserved in sequence from human to chicken. Cerebellin is highlyexpressed in cerebellum, weakly expressed in other areas of the brain,and possibly also expressed in adrenomedullary tissue (Satoh, F et al.,Journal of Endocrinology 154:27-34 (1997) which disclosure is herebyincorporated by reference in its entirety). Cerebellin is also expressedby the tumor tissues of adrenal tumor, ganglioneuroblastoma, andneuroblastoma (Satoh, F et al., Journal of Endocrinology 154:27-34(1997) which disclosure is hereby incorporated by reference in itsentirety). Cerebellin elicits epinephrine and norepinephrine release byhuman adrenomedullary cells (Albertin, G et al., Neuropeptides 34:7-11(2000) which disclosure is hereby incorporated by reference in itsentirety). Norepinephrine has been shown to up-regulate expression ofthe proinflammatory cytokine interferon gamma by the Th1 subset of CD4+T lymphocytes (Swanson, M A et al., J Immunol 166:232-240 (2001) whichdisclosure is hereby incorporated by reference in its entirety).Interferon gamma plays an important role in the development of cytolyticT lymphocytes (CTL). Recently, a precerebellin-like protein was shown tobe part of the acute phase response in rainbow trout (Gerwick, L et al.,Developmental and Comparative Immunology 24:597-600 (2000) whichdisclosure is hereby incorporated by reference in its entirety). Theinstant invention is based on the discovery that the CRPF polypeptidesoppose the proinflammatory action of cerebellin.

Thus, the invention is drawn to CRPF polypeptides, polynucleotidesencoding said CRPF polypeptides, vectors comprising said CRPFpolynucleotides, and cells recombinant for said CRPF polynucleotides, aswell as to pharmaceutical and physiologically acceptable compositionscomprising said CRPF polypeptides and methods of administering said CRPFpharmaceutical and physiologically acceptable compositions in order toreduce inflammation or to treat inflammation-related disorders. Assaysfor identifying antagonists of CRPF polypeptide function are also partof the invention.

In a first aspect, the invention features purified, isolated,artificially synthesized, or recombinant CRPF polypeptide that hasanti-inflammatory activity. In preferred embodiment, CRPF polypeptide isCRPF-1 polypeptide comprising the amino acid sequence: SHHH (SEQ IDNO:122). In other preferred embodiment, CRPF polypeptide is CRPF-2Apolypeptide comprising the amino acid sequence: AANSKVAFSAVRSTNH (SEQ IDNO:123). In other preferred embodiment, CRPF polypeptide is CRPF-2Bpolypeptide comprising the amino acid sequence AANSKVAFSAVR (SEQ IDNO:124). In other preferred embodiment, CRPF polypeptide is CRPF-2Cpolypeptide comprising the amino acid sequence STNH (SEQ ID NO:125). Inother preferred embodiment, CRPF polypeptide is CRPF-3 comprising theamino acid sequence SGSAKVAFSATRSTNH (SEQ ID NO:126).

In a second aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of said CRPF polypeptides described in thefirst aspect and, alternatively, a pharmaceutical or physiologicallyacceptable diluent.

In a third aspect, the invention features a method of reducinginflammation comprising providing or administering to individuals inneed of reducing inflammation said pharmaceutical or physiologicallyacceptable composition described in the second aspect. In preferredembodiment, said administration of said pharmaceutical orphysiologically acceptable composition is systemic or local.

In a fourth aspect, the invention features a method of preventing ortreating psoriasis comprising providing or administering to anindividual in need of such treatment said pharmaceutical orphysiologically acceptable composition described in the second aspect.

In a fifth aspect, the invention features a method of preventing ortreating atherosclerosis comprising providing or administering to anindividual in need of such treatment said pharmaceutical orphysiologically acceptable composition described in the second aspect.

In a sixth aspect, the invention features a method of preventing ortreating the inflammation associated with ischemic neurodegeneration inbrain tissue comprising providing or administering to an individual inneed of such treatment said pharmaceutical or physiologically acceptablecomposition described in the second aspect.

In a seventh aspect, the invention features a method of preventing ortreating the inflammation associated with stroke comprising providing oradministering to an individual in need of such treatment saidpharmaceutical or physiologically acceptable composition described inthe second aspect.

In an eighth aspect, the invention features a method of preventing ortreating the inflammation associated with myocardial infarctioncomprising providing or administering to an individual in need of suchtreatment said pharmaceutical or physiologically acceptable compositiondescribed in the second aspect.

In a ninth aspect, the invention features a method of preventing ortreating the inflammation associated with reperfusion injury comprisingproviding or administering to an individual in need of such treatmentsaid pharmaceutical or physiologically acceptable composition describedin the second aspect.

In a tenth aspect, the invention features a method of preventing ortreating the inflammation associated with rheumatoid arthritiscomprising providing or administering to an individual in need of suchtreatment said pharmaceutical or physiologically acceptable compositiondescribed in the second aspect.

In an eleventh aspect, the invention features a method of preventing ortreating the inflammation associated with inflammatory bowel diseasecomprising providing or administering to an individual in need of suchtreatment said pharmaceutical or physiologically acceptable compositiondescribed in the second aspect.

In a twelfth aspect, the invention features a method of preventing ortreating the inflammation associated with systemic lupus erythematosuscomprising providing or administering to an individual in need of suchtreatment said pharmaceutical or physiologically acceptable compositiondescribed in the second aspect.

In a thirteenth aspect, the invention features a method of preventing ortreating the inflammation associated with allergic asthma comprisingproviding or administering to an individual in need of such treatmentsaid pharmaceutical or physiologically acceptable composition describedin the second aspect.

In a fourteenth aspect, the invention features a method of preventing ortreating the inflammation associated with insulin dependent diabetesmellitus (Type 1 diabetes) comprising providing or administering to anindividual in need of such treatment said pharmaceutical orphysiologically acceptable composition described in the second aspect.

In a fifteenth aspect, the invention features a method of preventing ortreating the inflammation associated with septic shock comprisingproviding or administering to an individual in need of such treatmentsaid pharmaceutical or physiologically acceptable composition describedin the second aspect.

In a sixteenth aspect, the invention features a method of preventing ortreating the inflammation associated with multiple sclerosis comprisingproviding or administering to an individual in need of such treatmentsaid pharmaceutical or physiologically acceptable composition describedin the second aspect.

In a seventeenth aspect, the invention features a method of preventingor treating the inflammation associated with elevated cerebellin levelin blood or locally at a site of inflammation or other disorderdescribed herein comprising providing or administering to an individualin need of such treatment said pharmaceutical or physiologicallyacceptable composition described in the second aspect. Preferably themagnitude of said elevated cerebellin level is at least 10%, 20%, 30%,35%, 40%, 50%, 75%, 100%, or 500%. Preferably, cerebellin level iselevated at the site where said CRPF polypeptide is administered.

In an eighteenth aspect, the invention features a method of suppressingan in vivo Th1 immune response in an animal model of inflammopathologyor immune dysfunction comprising providing or administering to saidanimal said pharmaceutical or physiologically acceptable compositiondescribed in the second aspect. Preferably said animal is a mammal. Mostpreferably, said animal is a rodent.

In a nineteenth aspect, the invention provides for an antibody thatspecifically binds to said CRPF polypeptide described in the firstaspect. Further preferred is said antibody that neutralizes the capacityof said CRPF polypeptide to reduce inflammation. Further preferred issaid antibody that neutralizes the capacity of said CRPF polypeptide tobind to a ligand. Further preferred is said antibody that neutralizesthe capacity of said CRPF polypeptide to bind to its receptor.

In a twentieth aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of, said neutralizing antibody describedin the nineteenth aspect specific for said CRPF polypeptide described inthe first aspect and, alternatively, a pharmaceutical or physiologicallyacceptable diluent.

In a twenty-first aspect, the invention features a method of using saidantibody described in the nineteenth aspect specific for said CRPFpolypeptide described in the first aspect to measure the amount of saidCRPF polypeptide in a clinical sample in order to diagnose or stratifyan inflammation-related disorder. Preferably, said clinical sample isselected from the group consisting of blood, serum, plasma, urine,saliva, and lymphoid tissue. Preferably, said inflammation-relateddisorder is selected from but not restricted to those described herein.Reduced levels of said CRPF polypeptide are expected to be contributoryto said inflammation-related disorder.

In a twenty-second aspect, the invention features a method of using saidantibody described in the nineteenth aspect specific for said CRPFpolypeptide described in the first aspect to measure the amount of saidCRPF polypeptide in a clinical sample obtained from a cancer patient forthe purpose of characterizing said cancer. Preferably, said clinicalsample is selected from but not restricted to blood, serum, plasma,urine, saliva, and cancer tissue. Preferably, said method is selectedfrom but not restricted to ELISA or immunohistochemistry. Preferably,said cancer is selected from but not restricted to adrenal tumor,ganglioneuroblastoma, neuroblastoma, melanoma, squamous cell carcinomaof the skin, breast carcinoma, lung small-cell carcinoma, coloncarcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, prostaticcarcinoma, pancreatic carcinoma, osteosarcoma, uterine carcinoma,ovarian carcinoma, chondrosarcoma, endometrial cancer, testicularcarcinoma, renal carcinoma, hepatic carcinoma, lung non-small-cellcarcinoma, T cell acute lymphoblastic leukemia, B cell acutelymphoblastic leukemia, acute myeloid leukemia, chronic lymphocyticleukemia, or multiple myeloma. Elevated levels of said CRPF polypeptide(for example released by the tumor cells) are expected to becontributory to an impaired anti-tumor response.

In a twenty-third aspect, the invention features a method of treatingcancer characterized by elevated level of said CRPF polypeptidedescribed in the first aspect comprising providing or administering toan individual in need of such treatment said pharmaceutical orphysiologically acceptable composition described in the twentiethaspect. Neutralization of the capacity of said CRPF polypeptide suppressan inflammatory response is expected to facilitate the in vivoanti-tumor response. Preferably, said cancer is selected from but notrestricted to adrenal tumor, ganglioneuroblastoma, neuroblastoma,melanoma, squamous cell carcinoma of the skin, breast carcinoma, lungsmall-cell carcinoma, colon carcinoma, Hodgkin's lymphoma, non-Hodgkin'slymphoma, prostatic carcinoma, pancreatic carcinoma, osteosarcoma,uterine carcinoma, ovarian carcinoma, chondrosarcoma, endometrialcancer, testicular carcinoma, renal carcinoma, hepatic carcinoma, lungnon-small-cell carcinoma, T cell acute lymphoblastic leukemia, B cellacute lymphoblastic leukemia, acute myeloid leukemia, chroniclymphocytic leukemia, or multiple myeloma.

In a twenty-fourth aspect, the invention provides for a non-antibodycompound that specifically binds said CRPF polypeptide described in thefirst aspect but not to unrelated polypeptide. Preferred said compoundis selected from but not restricted to small molecular weight organic orinorganic compound, protein, peptide, carbohydrate, or lipid. Furtherpreferred is said compound that neutralizes the capacity of said CRPFpolypeptide to reduce inflammation.

Further preferred are methods of screening for one or more compoundscapable of neutralizing the capacity of said CRPF polypeptide fragmentto reduce inflammation. Said methods are well known to those skilled inthe art and include, but are not limited to: administering to a mousemodel of inflammopathology said CRPF polypeptide in the presence of saidantibody; administering to said mouse model said CRPF polypeptide in theabsence of said antibody; and determination of whether said antibodyabrogates the capacity of said CRPF polypeptide to reduce inflammationin said mouse model.

In a twenty-fifth aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of, said neutralizing compound of thetwenty-fourth aspect and, alternatively, a pharmaceutical orphysiologically acceptable diluent.

In a twenty-sixth aspect, the invention features a method of treating acancer patient having elevated levels of said CRPF polypeptide describedin the first aspect comprising providing or administering to anindividual in need of such treatment said pharmaceutical orphysiologically acceptable composition described in the twenty-fifthaspect. Preferably, methods for measuring the amount of said CRPFpolypeptide fragment include but are not restricted to said methodsdescribed in the twenty-second aspect. Neutralization of the capacity ofsaid CRPF polypeptide to suppress an inflammatory response is expectedto facilitate the in vivo anti-tumor response. Preferably, said canceris selected from but not restricted to adrenal tumor,ganglioneuroblastoma, neuroblastoma, melanoma, squamous cell carcinomaof the skin, breast carcinoma, lung small-cell carcinoma, coloncarcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, prostaticcarcinoma, pancreatic carcinoma, osteosarcoma, uterine carcinoma,ovarian carcinoma, chondrosarcoma, endometrial cancer, testicularcarcinoma, renal carcinoma, hepatic carcinoma, lung non-small-cellcarcinoma, T cell acute lymphoblastic leukemia, B cell acutelymphoblastic leukemia, acute myeloid leukemia, chronic lymphocyticleukemia, or multiple myeloma.

Uses of Antibodies

Antibodies of the present invention have uses that include, but are notlimited to, methods known in the art to purify, detect, and target thepolypeptides of the present invention including both in vitro and invivo diagnostic and therapeutic methods. For example, the antibodieshave use in immunoassays for qualitatively and quantitatively measuringlevels of antigen-bearing substances, including the polypeptides of thepresent invention, in biological samples (See, e.g., Harlow et al.,1988). The antibodies may also be used in therapeutic compositions forkilling cells expressing the protein or reducing the levels of theprotein in the body.

The invention further relates to antibodies that act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies that disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. These antibodies may act as agonists foreither all or less than all of the biological activities affected byligand-mediated receptor activation. The antibodies may be specified asagonists or antagonists for biological activities comprising specificactivities disclosed herein. The above antibody agonists can be madeusing methods known in the art. See e.g., WO 96/40281; U.S. Pat. No.5,811,097; Deng et al., (1998) Blood. 92(6):1981-1988; Chen et al.,(1998), Cancer Res. 58(16):3668-3678; Harrop et al., (1998), J. Immunol.161(4):1786-1794; Zhu, et al. (1998), Cancer Res. 58(15):3209-3214;Yoon, et al. (1998), J. Immunol. 160(7):3170-3179; Prat et al., (1998),J. Cell. Sci. 111(Pt2):237-247; Pitard et al., (1997), J. Immunol.Methods. 205(2):177-190; Liautard et al., (1997), Cytokine.9(4):233-241; Carlson et al., (1997), J. Biol. Chem.,272(17):11295-11301; Taryman, et al., (1995), Neuron. 14(4):755-762;Muller et al., (1998), Structure. 6(9):1153-1167; Bartunek et al.,(1996), Cytokine. 8(1):14-20.

As discussed above, antibodies of the polypeptides of the invention can,in turn, be utilized to generate anti-idiotypic antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art (see, e.g. Greenspan and Bona (1989), FASEB J.7(5):437-444 and Nissinoff, (1991), J. Immunol. 147(8): 2429-2438). Forexample, antibodies which bind to and competitively inhibit polypeptidemultimerization or binding of a polypeptide of the invention to ligandcan be used to generate anti-idiotypes that “mimic” the polypeptidemultimerization or binding domain and, as a consequence, bind to andneutralize polypeptide or its ligand. Such neutralization anti-idiotypicantibodies can be used to bind a polypeptide of the invention or to bindits ligands/receptors, and thereby block its biological activity.

Immunoaffinity Chromatography

Antibodies prepared as described herein are coupled to a support.Preferably, the antibodies are monoclonal antibodies, but polyclonalantibodies may also be used. The support may be any of those typicallyemployed in immunoaffinity chromatography, and the antibodies may becoupled to the support using any standard reagent. After coupling theantibody to the support, the support is contacted with a sample whichcontains a target polypeptide whose isolation, purification orenrichment is desired.

Thereafter, the support is washed with an appropriate wash solution, andthe specifically bound target polypeptide is eluted from the supportusing the high pH or low pH elution solutions typically employed inimmunoaffinity chromatography.

Expression of Genset Gene Products

Evaluation of Expression Levels and Patterns of GensetPolypeptide-Encoding mRNAs

The spatial and temporal expression patterns of GENSETpolypeptide-encoding mRNAs, as well as their expression levels, may bedetermined using any suitable method.

In one embodiment, expression levels and patterns of GENSETpolypeptide-encoding mRNAs is analyzed by solution hybridization withprobes (see, e.g., WO 97/05277). Briefly, an RNA complementary to themRNA of interest is labeled and derivatized with a capturable moiety,e.g., biotin. After hybridization in solution with mRNA isolated fromcells or tissues of interest, unhybridized probe is removed bydigestion, and the remaining, hybridized RNA is captured, e.g., on amicrotitration plate, and quantified.

In another embodiment, the GENSET polypeptide-encoding cDNAs, orfragments thereof, may also be tagged with nucleotide sequences for theserial analysis of gene expression (SAGE) (see, e.g., UK PatentApplication No. 2 305 241 A).

Quantitative analysis of GENSET gene expression may also be performedusing arrays. For example, quantitative analysis of gene expression maybe performed with GENSET polynucleotides, or fragments thereof in acomplementary DNA microarray as described by Schena et al. (1995)Science 270:467-470 and Schena et al. (1996), PNAS, 93(20):10614-10619,Pietu et al., (1996) Genome Research 6:492-503) or using any othermicroarray technology. Briefly, GENSET polypeptide-encoding cDNAs orfragments thereof are arrayed onto slides, and the arrays are hybridizedwith probes derived from mRNA of cells or tissues of interest. Followingwashing, the arrays are scanned using a fluorescence laser scanningdevice fitted with a custom filter set. Accurate differential expressionmeasurements are obtained by taking the average of the ratios of twoindependent hybridizations.

Alternatively, expression analysis of GENSET genes can be done throughhigh density nucleotide arrays as described by Lockhart et al., (1996)Nature Biotechnology 14: 1675-1680 and Sosnowski, et al., (1997) PNAS94:1119-1123. Oligonucleotides of 15-50 nucleotides corresponding tosequences of a GENSET polynucleotide or fragments thereof aresynthesized directly on the chip or synthesized and then addressed tothe chip. Labeled cDNA probes are synthesized from the appropriate mRNApopulation and then randomly fragmented to an average size of 50 to 100nucleotides. The probes are then hybridized to the chip. After washing,the label is detected and quantified. Comparative analysis of theintensity of the signal originating from cDNA probes on the same targetoligonucleotide in different cDNA samples indicates a differentialexpression of the GENSET polypeptide-encoding mRNA.

Uses of GENSET Gene Expression Data

Once the expression levels and patterns of a GENSET polypeptide-encodingmRNA has been determined, this information may be used to design GENSETgene specific markers for detection, identification, screening anddiagnostic purposes as well as to design DNA constructs with anexpression pattern similar to a GENSET gene expression pattern.

Detection of GENSET Polypeptide Expression and/or Biological Activity

The invention further relates to methods of detection of GENSETpolypeptide expression and/or biological activity in a biological sampleusing the polynucleotide and polypeptide sequences described herein.Such methods can be used, for example, as a screen for normal orabnormal GENSET polypeptide expression and/or biological activity and,thus, can be used diagnostically.

Detection of GENSET Polypeptides

The invention further relates to methods of detection of GENSETpolypeptides in a cell or sample. In certain embodiments, the presenceof polypeptides in a cell or sample is detected indirectly, by detectingthe presence of mRNA encoding the polypeptide. For example, a labeledpolynucleotide probe can be used in a method to detect a GENSETpolypeptide-encoding mRNA, wherein the presence of the mRNA isindicative of the expression of the GENSET polypeptide-encoding gene.

Consequently, the invention comprises methods for detecting the presenceof a polynucleotide of the invention in a sample, the method comprisingbringing into contact said sample and a nucleic acid probe or aplurality of nucleic acid probes which hybridize to the polynucleotide,and detecting the hybrid complex formed between said probe or saidplurality of probes and said polynucleotide. In certain embodiments, theprobe or probes are labeled or are immobilized on a substrate.

In another embodiment, the polynucleotide is detected using anamplification reaction, wherein the sample is contacted withamplification reaction reagents, an amplification reaction is performedto synthesize amplification products containing said region of saidselected nucleotide sequence; and the amplification products aredetected. In a preferred embodiment, when the polynucleotide to beamplified is a RNA molecule, preliminary reverse transcription andsynthesis of a second cDNA strand are first performed in order toprovide a DNA template to be amplified.

Alternatively, a method of detecting GENSET polypeptide expression in atest sample can be accomplished using any product which binds to aGENSET polypeptide of the present invention or portion thereof. Suchproducts may be antibodies, binding fragments of antibodies,polypeptides able to bind specifically to GENSET polypeptides orfragments thereof, including GENSET polypeptide agonists andantagonists. Detection of specific binding to the antibody indicates thepresence of a GENSET polypeptide in the sample (e.g., ELISA).

Consequently, the invention is also directed to a method for detectingspecifically the presence of a GENSET polypeptide in a biologicalsample, said method comprising bringing into contact the biologicalsample with a product able to bind to a polypeptide of the invention orfragments thereof; allowing the product to bind to the polypeptide toform a complex; and detecting the complex. In a preferred embodiment,the product is an antibody, e.g., an antibody that is immobilized on asubstrate.

The present invention also relates to kits that can be used in thedetection of GENSET polypeptide-encoding gene expression products, e.g.containing a compound that specifically binds a GENSET polypeptide (e.g.binding proteins, antibodies or binding fragments thereof (e.g. F(ab′)2fragments) or a GENSET polypeptide-encoding mRNA (e.g. a complementaryprobe or primer), disposed within a container means. The kit can furthercomprise ancillary reagents, including buffers and the like.

Detection of GENSET Polypeptide Biological Activity

The invention further includes methods of detecting specifically aGENSET polypeptide biological activity, and to identify compoundscapable of modulating the activity of a GENSET polypeptide. Assessingthe GENSET polypeptide biological activity may be performed by thedetection of a change in any cellular property associated with theGENSET polypeptide, using a variety of techniques, including thosedescribed herein. To identify modulators of the polypeptides, a controlis preferably used. For example, a control sample includes all of thesame reagents but lacks the compound or agent being assessed; it istreated in the same manner as the test sample.

The present invention also relates to kits that can be used in thedetection of GENSET polypeptide biological activity, e.g., includingsubstrates for GENSET polypeptides, GENSET-binding compounds, antibodiesto GENSET polypeptides, etc., disposed within a container means. The kitcan further comprise ancillary reagents, including buffers and the like.

Identification of a Specific Context of GENSET Polypeptide-Encoding GeneExpression

When the expression pattern of a GENSET polypeptide-encoding mRNA showsthat a GENSET polypeptide-encoding gene is specifically expressed in agiven context, probes and primers specific for this gene as well asantibodies binding to the GENSET polypeptide-encoding polynucleotide maythen be used as markers for the specific context. Examples of specificcontexts are: specific expression in a given tissue/cell or tissue/celltype, expression at a given stage of development of a process such asembryo development or disease development, expression in response to aparticular compound or drug, or specific expression in a givenorganelle. Such primers, probes, and antibodies are useful commerciallyto identify tissues/cells/organelles of unknown origin, for example,forensic samples, differentiated tumor tissue that has metastasized toforeign bodily sites, or to differentiate different tissue types in atissue cross-section.

Determination of tissue/cell/organelle identity is based on methods thatdetect the presence or absence of the mRNA (or corresponding cDNA orprotein) in a tissue/cell sample using methods well known in the art(e.g., hybridization, PCR based methods, immunoassays, immunochemistry,ELISA). Therefore, the invention encompasses uses of thepolynucleotides, polypeptides, and antibodies of the invention as tissuemarkers.

Identification of Tissue Tapes or Cell Species by Means of LabeledTissue Specific Antibodies

Identification of specific tissues is accomplished by the visualizationof tissue specific antigens by means of antibody preparations which areconjugated, directly (e.g., green fluorescent protein) or indirectly toa detectable marker. Selected labeled antibody species bind to theirspecific antigen binding partner in tissue sections, cell suspensions,or in extracts of soluble proteins from a tissue sample to provide apattern for qualitative or semi-qualitative interpretation.

A. Immunohistochemical Techniques

Purified, high-titer antibodies, prepared as described above, areconjugated to a detectable marker, as described, for example, byFudenberg, (1980) Chap. 26 in: Basic 503 Clinical Immunology, 3rd Ed.Lange, Los Altos, Calif. or Rose et al., (1980) Chap. 12 in: Methods inImmunodiagnosis, 2d Ed. John Wiley 503 Sons, New York.

Antibodies can be labeled using any suitable method, e.g. fluorescentlabels, enzymes (e.g. horseradish peroxidase), ferritin (for detectionusing EM), or radiolabeling. In one embodiment, cryostat sections of theunknown tissue and known control are mounted and each slide covered withdifferent dilutions of the antibody preparation. Following incubation,excess fluid is blotted away, and the marker detected. The antigen foundin the tissues by the above procedure can be quantified by measuring theintensity of color or fluorescence on the tissue section, andcalibrating that signal using appropriate standards.

B. Identification of Tissue Specific Soluble Proteins

The visualization of tissue specific proteins and identification ofunknown tissues from that procedure is carried out using the labeledantibody reagents and detection strategy as described forimmunohistochemistry; however the sample is prepared according to anelectrophoretic technique to distribute the proteins extracted from thetissue in an orderly array on the basis of molecular weight fordetection, e.g. by western blotting. See, e.g., Davis et al., BasicMethods in Molecular Biology, ed., Elsevier Press, NY (1986), Section19-3.

Screening and Diagnosis of Abnormal GENSET Polypeptide Expression and/orBiological Activity

Moreover, antibodies and/or primers or probes specific for GENSETpolypeptide expression may also be used to identify abnormal GENSETpolypeptide expression and/or biological activity, and subsequently toscreen and/or diagnose disorders associated with abnormal GENSETpolypeptide expression. For example, a particular disease may resultfrom lack of expression, over-expression, or under-expression of aGENSET polypeptide-encoding mRNA. By comparing mRNA expression patternsand quantities in samples taken from healthy individuals with those fromindividuals suffering from a particular disorder, genes responsible forthis disorder may be identified.

Screening for Specific Disorders

The present invention also relates to methods and uses of GENSETpolypeptides for identifying individuals having elevated or reducedlevels of GENSET polypeptides, which individuals are likely to benefitfrom therapies to suppress or enhance GENSET polypeptide-encoding geneexpression, respectively. One example of such methods and uses comprisesdetecting the presence in a biological sample of a GENSETpolypeptide-encoding gene product (mRNA or protein); comparing theamount of the GENSET polypeptide-encoding gene product present in saidsample with that of a control sample; and determining whether the samplehas a reduced or elevated level of GENSET gene expression compared tothe control sample.

A biological sample from a subject affected by, or at risk ofdeveloping, any disease or condition associated with a GENSETpolypeptide can be screened for the presence of increased or decreasedlevels of GENSET gene product, relative to a normal population (standardor control), with an increased or decreased level of the GENSETpolypeptide relative to the normal population being indicative ofpredisposition to or a present indication of the disease or condition,or any symptom associated with the disease or condition. Suchindividuals would be candidates for therapies, e.g., treatment withpharmaceutical compositions comprising the GENSET polypeptide, apolynucleotide encoding the GENSET polypeptide, or any other compoundthat affects the expression or activity of the GENSET polypeptide.Generally, the identification of elevated levels of the GENSETpolypeptide in a patient would be indicative of an individual that wouldbenefit from treatment with agents that suppress GENSET polypeptideexpression or activity, and the identification of low levels of theGENSET polypeptide in a patient would be indicative of an individualthat would benefit from agents that induce GENSET expression oractivity.

Biological samples suitable for use in this method include anybiological fluids, including, but not limited to, blood, saliva, milk,and urine, as well as tissue samples such as biopsies. Cell cultures orcell extracts derived, for example, from tissue biopsies can also beused. In preferred embodiments, the biological sample is taken fromanimals presenting any symptom associated with any disease or conditionassociated with a GENSET gene product. In accordance with this method,the presence in the sample of altered (e.g. increased or decreased)levels of the GENSET product indicates that the subject is predisposedto the disease or condition.

The diagnostic methodologies described herein are applicable to bothhumans and non-human mammals.

Detection of GENSET Gene Mutations

The invention also encompasses methods and uses of GENSETpolynucleotides to detect mutations in GENSET polynucleotides of theinvention. When the mutation was proven to be associated with a disease,the detection of such mutations may be used for screening and diagnosispurposes.

In one embodiment, an oligonucleotide probe matrix may advantageously beused to detect mutations occurring in GENSET genes and preferably intheir regulatory regions. For this particular purpose, probes arespecifically designed to have a nucleotide sequence allowing theirhybridization to the genes that carry known mutations (either bydeletion, insertion or substitution of one or several nucleotides). Byknown mutations, it is meant, mutations on the GENSET genes that havebeen identified according, for example to the technique used by Huang etal., (1996) Cancer Res 56(5):1137-1141 or Samson et al., (1996) Nature,382(6593):722-725.

Another technique that is used to detect mutations in GENSET genes isthe use of a high-density DNA array. Each oligonucleotide probeconstituting a unit element of the high density DNA array is designed tomatch a specific subsequence of a GENSET genomic DNA or cDNA. Thus, anarray consisting of oligonucleotides complementary to subsequences ofthe target gene sequence is used to determine the identity of the targetsequence with the wild gene sequence, measure its amount, and detectdifferences between the target sequence and the reference wild genesequence of the GENSET gene. In one such embodiment, a 4 L tiled arrayis used (see, e.g., Chee et al., (1996) Science. 274:610-614).

Construction of DNA Constructs with a GENSET Gene Expression Pattern

In addition, characterization of the spatial and temporal expressionpatterns and expression levels of GENSET polypeptide-encoding mRNAs isalso useful for constructing expression vectors capable of producing adesired level of gene product in a desired spatial or temporal manner,as discussed below.

DNA Constructs that Direct Temporal and Spatial GENSET Gene Expressionin Recombinant Cell Hosts and in Transgenic Animals.

In order to study the physiological and phenotypic consequences of alack of synthesis of a GENSET polypeptide, both at the cellular leveland at the multi-cellular organism level, the invention also encompassesDNA constructs and recombinant vectors enabling a conditional expressionof a specific allele of a GENSET polypeptide-encoding genomic sequenceor cDNA, including alleles that include substitutions, deletions, oradditions of one or more bases within the sequence.

In one embodiment, a DNA construct is used that is based on thetetracycline resistance operon tet from E. coli transposon Tn10 forcontrolling the GENSET gene expression, such as described by Gossen etal., (1992) PNAS 89:5547-5551; Gossen et al., (1995) Science268:1766-1769; and Furth P. A. et al. (1994) PNAS 91:9302-9306.

In another embodiment, a DNA construct is used that comprises, from5′-end to 3′-end: a first nucleotide sequence that is found in theGENSET polypeptide-encoding genomic sequence; a nucleotide sequencecomprising a positive selection marker (e.g. neo); and a secondnucleotide sequence that is found in the GENSET polypeptide-encodinggenomic sequence, and is located downstream of the first GENSETnucleotide sequence. In a preferred embodiment, the construct alsocomprises a negative selection marker (e.g. thymidine kinase,hygromycine beta, hprt, Diphtheria toxin A fragment) located upstream ofthe first GENSET nucleotide sequence or downstream from the secondGENSET nucleotide sequence (see, e.g., Thomas et al. (1986), Cell.44:419-428; Te Riele et al. (1990), Nature. 348:649-651; Van der Lugt etal. (1991), Gene 105:263-267; Reid et al., (1990) PNAS 87:4299-4303;Nada et al., (1993) Cell 73:1125-1135; Yagi, T., et al. (1990), PNAS87:9918-992; Thomas et al. (1986; 1987), Mansour et al. (1988) andKoller et al., (1992) Annu. Rev. Immunol. 10:705-730).

In another embodiment, vectors are used that involve the use of theCre-loxP system (Hoess et al., (1986) Nucleic Acids Res. 14:2287-2300).The Cre-loxP system used in combination with a homologous recombinationtechnique has been described by Gu H. et al., (1993) Cell 73:1155-1164and Gu H. et al., (1994) Science 265:103-106. Briefly, a nucleotidesequence of interest to be inserted in a targeted location of the genomeharbors at least two loxP sites in the same orientation and located atthe respective ends of a nucleotide sequence to be excised from therecombinant genome. The excision event requires the presence of therecombinase (Cre) enzyme within the nucleus of the recombinant cellhost, which may be supplied in any of a number of ways, e.g., byincubating the recombinant cell hosts in a culture medium containingthis enzyme, by injecting the Cre enzyme directly into the desired cell,by lipofection of the enzyme into the cells, or by otherwisetransfecting the cell with a Cre coding sequence under the control of anappropriate promoter (see, e.g., Baubonis et al (1993) Nucleic AcidsRes. 21(9):2025-9); Araki et al., (1995) PNAS 92(1): 160-4; Gu et al.(1993); Sauer et al., (1988) PNAS 85:5166-5170; Gu et al. (1994); Zou,et al, (1994) Curr. Biol. 4:1099-1103; Anton and Graham, (1995), J.Virol., 69: 4600-4606; and Kanegae et al., (1995) Nucl. Acids Res.23:3816-3821).

The DNA constructs described above may be used to introduce a desirednucleotide sequence of the invention, preferably a GENSET genomicsequence or a GENSET cDNA sequence, and most preferably an altered copyof a GENSET genomic or cDNA sequence, within a predetermined location ofthe targeted genome, leading either to the generation of an altered copyof a targeted gene (knock-out homologous recombination) or to thereplacement of a copy of the targeted gene by another copy sufficientlyhomologous to allow an homologous recombination event to occur (knock-inhomologous recombination).

Modifying GENSET Polypoptide Expression and/or Biological Activity

Modifying endogenous GENSET expression and/or biological activity isexpressly contemplated by the present invention.

Screening for Compounds that Modulate GENSET Expression and/orBiological Activity

The present invention further relates to compounds able to modulateGENSET expression and/or biological activity and methods to use thesecompounds. Such compounds may interact with the regulatory sequences ofGENSET genes or they may interact with GENSET polypeptides directly orindirectly.

Compounds Interacting With GENSET Regulatory Sequences

The present invention also concerns a method for screening substances ormolecules that are able to interact with the regulatory sequences of aGENSET gene, such as for example promoter or enhancer sequences inuntranscribed regions of the genomic DNA, as determined using anytechniques known to those skilled in the art, or such as regulatorysequences located in untranslated regions of GENSET mRNA.

Sequences within untranscribed or untranslated regions ofpolynucleotides of the invention may be identified by comparison todatabases containing known regulatory sequence such as transcriptionstart sites, transcription factor binding sites, promoter sequences,enhancer sequences, 5′UTR and 3′UTR elements (Pesole et al., (2000)Nucleic Acids Res, 28(1):193-196;http:/igs-server.cnrs-mrs.fr/˜gauthere/UTR/index.html). Alternatively,the regulatory sequences of interest may be identified throughconventional mutagenesis or deletion analyses of reporter plasmids.

Following the identification of potential GENSET regulatory sequences,proteins which interact with these regulatory sequences may beidentified as described below.

Any of a number of methods can be used to identify molecules capable ofinteracting with the regulatory sequence of a GENSET gene, such as gelretardation assays (see, e.g., Fried and Crothers, (1981) Nucleic AcidsRes. 9:6505-6525; Garner and Revzin, (1981) Nucleic Acids Res9:3047-3060; and Dent and Latchman (1993) The DNA mobility shift assay.In: Transcription Factors: A Practical Approach (Latchman DS, ed.) pp:1-26, Oxford: IRL Press). Nucleic acids encoding proteins which are ableto interact with the promoter sequence of a GENSET gene may also beidentified by using a one-hybrid system (e.g., the Matchmaker One-HybridSystem kit from Clontech (Catalog Ref. no. K1603-1)).

Ligands Interacting with Genset Polypeptides

For the purpose of the present invention, a ligand means a molecule,such as a protein, a peptide, an antibody or any synthetic chemicalcompound capable of binding to a GENSET protein or one of its fragmentsor variants or to modulate the expression of the polynucleotide codingfor GENSET or a fragment or variant thereof.

In one embodiment, a biological sample or a defined molecule (e.g. amolecule generated through combinatorial chemistry) to be tested as aputative ligand of a GENSET protein is brought into contact with thepurified GENSET protein, in order to determine if a complex is formedbetween this protein and a component of the sample or the definedmolecule. The interaction between such molecules and the protein can beassessed in any way, e.g., using microdialysis coupled to HPLC, usingaffinity capillary electrophoresis, etc. (see, e.g., Wang, et al.(1997), Chromatographia, 44: 205-208; Bush et al., (1997), J.Chromatogr., 777:311-328).

Any type of compound can be tested, using any method, for interactionwith a GENSET polypeptide or polynucleotide in the present methods,including, but not limited to, peptides, drugs, fatty acids,lipoproteins, or small molecules, and may be obtained from any source.For example, the molecule to be tested is labeled with a detectablelabel, such as a fluorescent, radioactive, or enzymatic tag and placedin contact with immobilized GENSET protein, or a fragment thereof underconditions which permit specific binding to occur. After removal ofnon-specifically bound molecules, bound molecules are detected usingappropriate means.

A. Candidate Ligands Obtained from Random Peptide Libraries

In a particular embodiment of the screening method, the putative ligandis the expression product of a DNA insert contained in a phage vector,e.g. from a random peptide phage library comprising peptides of from 8to 20 amino acids in length (Parmley and Smith (1988) Gene 73:305-318;Oldenburg et al. (1992) PNAS 89:5393-5397; Valadon et al. (1996) J. Mol.Biol., 261:11-22; Lucas (1994) In: Development and Clinical Uses ofHaempophilus b Conjugate; Westerink (1995) PNAS 92:4021-4025; Felici(1991) J. Mol. Biol., 222:301-310).

Once the ligand library in recombinant phages has been constructed, thephage population is brought into contact with the immobilized GENSETprotein, and, following washing, the phages that bind specifically tothe GENSET protein are either eluted by a buffer (acid pH) orimmunoprecipitated using an antibody specific to the GENSET protein. Theisolated phage is subsequently amplified by an over-infection ofbacteria (for example E. coli). The selection step may be repeatedseveral times, preferably 2-4 times, in order to select the morespecific recombinant phage clones.

B. Candidate Ligands Obtained by Competition Experiments.

Alternatively, peptides, drugs or small molecules which bind topolypeptides of the present invention may be identified in competitionexperiments. In one such assay, a GENSET protein, or a fragment thereof,is immobilized to a surface, such as a plastic plate. Increasing amountsof the peptides, drugs or small molecules are placed in contact with theimmobilized GENSET protein, or a fragment thereof, in the presence of adetectable labeled known GENSET protein ligand. For example, the GENSETligand may be detectably labeled with a fluorescent, radioactive, orenzymatic tag. The ability of the test molecule to bind the GENSETprotein, or a fragment thereof, is determined by measuring the amount ofdetectably labeled known ligand bound in the presence of the testmolecule. A decrease in the amount of known ligand bound to the GENSETprotein, or a fragment thereof, when the test molecule is presentindicated that the test molecule is able to bind to the GENSET protein,or a fragment thereof.

C. Candidate Ligands Obtained by Affinity Chromatography.

Proteins or other molecules interacting with a polypeptide of thepresent invention can also be found using affinity columns which containthe GENSET protein, or a fragment thereof. The GENSET protein, or afragment thereof, may be attached to the column using conventionaltechniques including chemical coupling to a suitable column matrix (e.g.agarose, Affi Gel®, etc.). In some embodiments of this method, theaffinity column contains chimeric proteins in which the GENSET protein,or a fragment thereof, is fused to glutathion S transferase (GST). Amixture of cellular proteins or pool of expressed proteins as describedabove is applied to the affinity column. Proteins or other moleculesinteracting with the GENSET protein, or a fragment thereof, attached tothe column can then be isolated and analyzed, e.g., on 2-Delectrophoresis gel as described in Ramunsen et al., (1997),Electrophoresis, 18: 588-598. Alternatively, the proteins retained onthe affinity column can be purified by electrophoresis-based methods andsequenced. The same method can be used to isolate antibodies, to screenphage display products, or to screen phage display human antibodies.

D. Candidate Ligands Obtained by Optical Biosensor Methods

Proteins interacting with a polypeptide of the present invention, canalso be screened by using an Optical Biosensor (see, e.g., Edwards andLeatherbarrow (1997) Anal. Bioch. 246:1-6; Szabo et al., (1995) CurrOpin Struct Biol 5, 699-705. This technique permits the detection ofinteractions between molecules in real time, without the need of labeledmolecules. This technique is based on the surface plasmon resonance(SPR) phenomenon. In one embodiment, a GENSET polypeptide, or fragmentthereof, is attached to a surface (such as a carboxymethyl dextranmatrix) comprising one side of a cell through which flows the candidatemolecule to be assayed. A light beam is directed towards the side of thesurface that does not contain the sample to be tested and is reflectedby said surface. The binding of a candidate ligand molecules cause achange in the refraction index on the surface, which change is detectedas a change in the SPR signal. This technique may also be performed byimmobilizing eukaryotic or prokaryotic cells or lipid vesiclesexhibiting an endogenous or a recombinantly expressed GENSET protein attheir surface.

E. Candidate Ligands Obtained Through a Two-Hybrid Screening Assay.

The yeast two-hybrid system is designed to study protein-proteininteractions in vivo (Fields and Song, 1989; U.S. Pat. Nos. 5,667,973and 5,283,173), and relies upon the fusion of a bait protein to the DNAbinding domain of the yeast Gal4 protein. The general procedure oflibrary screening by the two-hybrid assay may be performed as describedby Harper et al., (1993), Cell 75:805-816; Cho et al., (1998) PNAS95(7):3752-3757; Fromont-Racine et al., (1997) Nature Genetics16(3):277-282. In typical embodiments, the bait protein comprises apolypeptide of the present invention, and the “prey” comprises a humancDNA library constructed such that the human cDNA insert is fused to anucleotide sequence in the vector that encodes the transcriptionaldomain of the GAL4 protein. In another embodiment, interaction betweenthe GENSET polypeptide or a fragment or variant thereof with cellularproteins is assessed using the Matchmaker Two Hybrid System 2 (CatalogNo. K1604-1, Clontech).

Compounds Modulating GENSET Biological Activity

Another method of screening for compounds that modulate GENSETexpression and/or biological activity is by measuring the effects oftest compounds on specific biological activity, e.g. a GENSET biologicalactivity in a host cell or in an in vitro assay. A GENSET biologicalactivity can include any of the activities, functions, or propertiesdescribed herein for any GENSET polypeptide or polynucleotide. In oneembodiment, a nucleic acid construct encoding a GENSET polypeptide isintroduced into a host cell, and the host cell is maintained underconditions appropriate for expression of the encoded GENSET polypeptide,whereby the nucleic acid is expressed. The host cell is then contactedwith a test agent, wherein a detection of a change in any GENSETpolypeptide-associated property in the presence of the agent indicatesthat the agent alters GENSET biological activity. In a particularembodiment, the invention relates to a method of identifying an agentwhich is an activator of GENSET biological activity, wherein detectionof an increase of any GENSET polypeptide-associated property in thepresence of the agent indicates that the agent activates GENSETbiological activity. In another particular embodiment, the inventionrelates to a method of identifying an agent which is an inhibitor ofGENSET biological activity, wherein detection of a decrease of anyGENSET polypeptide-associated property in the presence of the agentindicates that the agent inhibits GENSET biological activity. In anotherparticular embodiment, a high throughput screen is used to identifyagents that activate (enhance) or inhibit GENSET biological activity(see, e.g., WO 98/45438).

Methods of Screening for Compounds Modulating GENSET Gene Expressionand/or Activity

The present invention also relates to methods of screening compounds fortheir ability to modulate (e.g. increase or inhibit) the activity orexpression of GENSET polypeptides and polynucleotides. Morespecifically, the present invention relates to methods of testingcompounds for their ability either to increase or to decrease expressionor activity of GENSET polypeptides and polynucleotides. The assays areperformed in vitro or in vivo.

In Vitro Methods

In vitro, cells expressing GENSET polypeptides, or capable of expressingGENSET polypeptides, are incubated in the presence and absence of thetest compound. By determining the level of GENSET expression in thepresence of the test compound or the level of GENSET biological activityin the presence of the test compound, compounds can be identified thatsuppress or enhance GENSET expression or activity. Alternatively,constructs comprising a GENSET regulatory sequence operably linked to areporter gene (e.g. luciferase, chloramphenicol acetyl transferase,LacZ, green fluorescent protein, etc.) can be introduced into host cellsand the effect of the test compounds on expression of the reporter genedetected. Consequently, the present invention encompasses a method forscreening molecules that modulate the expression of a GENSET gene, saidscreening method comprising cultivating a prokaryotic or a eukaryoticcell that has been transfected with a nucleotide sequence encodingeither a GENSET polypeptide, placed under the control of its ownpromoter, or a detectable polypeptide, placed under the control of aGENSET 5′ regulatory region; bringing into contact the cultivated cellwith a molecule to be tested; and quantifying the expression of theGENSET or detectable polypeptide in the presence of the molecule. Themethod can also be performed using fragments, variants, or derivativesof any of the GENSET polypeptides or 5′ regulatory regions.

The quantification of the expression of a GENSET polypeptide may berealized either at the mRNA level (using for example Northern blots,RT-PCR, preferably quantitative RT-PCR with primers and probes specificfor the GENSET mRNA of interest) or at the protein level (usingpolyclonal or monoclonal antibodies in immunoassays such as ELISA or RIAassays, Western blots, or immunochemistry).

In a further embodiment, the GENSET 5′ regulatory region includes the5′UTR region of a GENSET polynucleotide sequence, and/or a promotersequence which is endogenous or exogenous with respect to the GENSET5′UTR sequence.

The invention further relates to a method for the production of apharmaceutical composition comprising identifying a molecule thatmodulates the expression of a GENSET gene using any of theherein-described methods, and combining the identified molecule with aphysiologically acceptable carrier.

Kits for the screening candidate substances for the ability to modulatethe expression of a GENSET gene. Preferably, such kits comprise arecombinant vector comprising a GENSET 5′ regulatory region or aregulatory active fragment or a variant thereof, operably linked to apolynucleotide encoding a detectable protein or a GENSET protein or afragment or a variant thereof.

Another object of the present invention comprises methods and kits forthe screening of candidate substances that interact with a GENSETpolypeptide, fragments or variants thereof. By their capacity to bindcovalently or non-covalently to a GENSET protein, fragments or variantsthereof, these substances or molecules may be advantageously used bothin vitro and in vivo.

In vitro, said interacting molecules may be used as detection means inorder to identify the presence of a GENSET protein in a sample,preferably a biological sample.

In one embodiment, a method is provided for the screening of a candidatesubstance, the method comprising providing a GENSET protein; bringinginto contact the protein with the candidate substance; and determiningwhether a complex is formed between the polypeptide or fragment and thecandidate substance.

The invention further relates to a method for the production of apharmaceutical composition comprising identifying a substance thatinteracts with a GENSET polypeptide using any of the herein-describedmethods, fragments or variants thereof and furthermore mixing theidentified substance with a physiologically acceptable carrier.

The invention further concerns a kit for the screening of a candidatesubstance interacting with the GENSET polypeptide, wherein said kitcomprises a GENSET polypeptide, and optionally means to detect a complexformed between the polypeptide and the candidate substance. In oneembodiment, the detection means comprises a monoclonal or polyclonalantibody binding to said GENSET protein or fragment or variant thereof.

In Vivo Methods

Compounds that suppress or enhance GENSET gene expression can also beidentified using in vivo screens. In a typical assay, a test compound isadministered (e.g. intravenously, intraperitoneally, intramuscularly,orally, or otherwise) to an animal, at a variety of dose levels, and theeffect of the compound on GENSET gene expression is determined bycomparing the levels of the mRNA or protein encoded by the gene intissues known to express the gene of interest, e.g., using Northernblots, immunoassays, PCR, etc. Suitable test animals include, but arenot limited to, rodents (e.g., mice and rats), primates, and rabbits.Humanized mice can also be used, that is mice in which the endogenousmouse protein is ablated (knocked out) and the homologous human proteinintroduced using standard transgenic approaches. Such mice thus expressonly the human form of a protein. Humanized mice expressing only thehuman GENSET polypeptide can be used to study in vivo responses topotential agents regulating GENSET protein or mRNA levels. Suchtransgenic animals are useful for dissecting the biochemical andphysiological steps of disease, and for development of therapies fordisease intervention (see, e.g., Loring, et al, 1996).

In addition, the detection of any change in any GENSET gene-associatedbehavior or characteristic of an animal following the administration ofa compound can also be used as an indication that the compound modulatesthe expression or activity of the gene.

Uses for Compounds Modulating GENSET Expression and/or BiologicalActivity

Using in vivo (or in vitro) systems, it may be possible to identifycompounds that exert a tissue specific effect, for example, thatincrease GENSET expression or activity in one or more particular tissuesof interest, such as the adrenal gland, bone marrow, brain, cerebellum,colon, fetal brain, fetal kidney, fetal liver, heart, hypertrophicprostate, kidney, liver, lung, lymph ganglia, lymphocytes, muscle,ovary, pancreas, pituitary gland, placenta, prostate, salivary gland,spinal cord, spleen, stomach, intestine, substantia nigra, testis,thyroid, umbilical cord, or uterus. Screening procedures such as thosedescribed above are also useful for identifying agents for theirpotential use in pharmacological intervention strategies. Agents thatenhance GENSET gene expression or stimulate its activity may thus beused to induce any phenotype associated with a GENSET gene, or to treatdisorders resulting from a deficiency of a GENSET polypeptide activityor expression. Compounds that suppress GENSET polypeptide expression orinhibit its activity can be used to treat any disease or conditionassociated with increased or deleterious GENSET polypeptide activity orexpression.

Also encompassed by the present invention is an agent which interactswith a GENSET gene or polypeptide directly or indirectly, and inhibitsor enhances GENSET polypeptide expression and/or function. In oneembodiment, the agent is an inhibitor which interferes with a GENSETpolypeptide directly (e.g., by binding the GENSET polypeptide) orindirectly (e.g., by blocking the ability of the GENSET polypeptide tohave a GENSET biological activity). In a particular embodiment, aninhibitor of a GENSET protein is an antibody specific for the GENSETprotein or a functional portion of the GENSET protein. Alternatively,the inhibitor can be an agent other than an antibody (e.g., smallorganic molecule, protein or peptide) which binds the GENSET polypeptideand blocks its activity. For example, the inhibitor can be an agentwhich mimics the GENSET polypeptide structurally, but lacks its function(e.g. a dominant negative form of the protein). Alternatively, it can bean agent which binds to or interacts with a molecule which the GENSETpolypeptide normally binds to or interacts with, thus blocking theGENSET polypeptide from doing so and preventing it from exerting theeffects it would normally exert.

In another embodiment, the agent is an enhancer (activator) of a GENSETpolypeptide which increases the activity of the GENSET polypeptide(increases the effect of a given amount or level of GENSET polypeptide),increases the length of time it is effective (by preventing itsdegradation or otherwise prolonging the time during which it is active)or both either directly or indirectly. For example, GENSETpolynucleotides and polypeptides can be used to identify drugs whichincrease or decrease the ability of GENSET polypeptides to induce GENSETbiological activity, which drugs are useful for the treatment orprevention of any disease or condition associated with a GENSETbiological activity.

Thus the present invention relates to a method of inhibiting (partiallyor completely) a GENSET biological activity in a mammal (e.g., a human),the method comprising administering to the mammal an effective amount ofan inhibitor of a GENSET polypeptide or polynucleotide. The inventionalso relates to a method of enhancing a GENSET biological activity in amammal, the method comprising administering to the mammal an effectiveamount of an enhancer of a GENSET polypeptide or polynucleotide.

Inhibiting GENSET Gene Expression

GENSET gene expression can be inhibited, e.g., for therapeuticapplications, in any of a large number of ways, including by using anantisense tool or a triple helix tool that inhibits the expression ofthe corresponding GENSET gene.

Antisense Approach

In antisense approaches, DNA and/or RNA sequences complementary to anmRNA are hybridized to the mRNA intracellularly, thereby blocking theexpression of the protein encoded by the mRNA. Preferred methods forusing antisense polynucleotide according to the present invention arethe procedures described by Sczakiel et al., (1995) Trends Microbiol.3(6):213-217; Green et al., (1986) Ann. Rev. Biochem. 55:569-597; Izantand Weintraub, (1984) Cell 36(4):1007-15; Liu et al. (1994) PNAS 91:4528-4262; Eckner et al., (1991) EMBO J. 10:3513-3522).

Preferably, the antisense tools are chosen among the polynucleotides(15-200 bp long) that are complementary to GENSET mRNA, more preferablyto the 5′ end (e.g. the translation initiation codon ATG) or to asplicing donor or acceptor site of the GENSET mRNA. In anotherembodiment, a combination of different antisense polynucleotidescomplementary to different parts of the desired targeted gene are used.The antisense molecules can be prepared in any way, including byreversing the orientation of a coding region of a GENSET gene in a cell,or by synthesizing an oligonucleotide in vitro and administering it tothe cell.

Any antisense sequence complementary to any portion of any of theherein-described polynucleotides can be used, and can involve any numberof modifications to the backbone, linkages, or bases, a large number ofwhich are known in the art. Suitable modifications, other forms ofantisense molecules, and the preparation thereof, are taught, interalia, in The Concise Encyclopedia Of Polymer Science And Engineering,pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990; Englischet al., Angewandte Chemie, International Edition (1991), 30, 613;Sanghvi, et al., eds., (1993) Antisense Research and Applications, CRCPress, Boca Raton; U.S. Pat. No. 6,242,590; WO94/23026, WO 96/31523; WO92/18522; European Patent Application No. 0 572 287 A2; WO 92/19732;Letsinger et al., PNAS (1989) 86: 6553-6556; Manoharan et al., Bioorg.Med. Chem. Let. (1994) 4:1053-1060; Manoharan et al., Ann. N.Y. Acad.Sci. (1992) 660:306-309; Manoharan et al., Bioorg. Med. Chem. Let.(1993) 3:2765-2770; Oberhauser et al., Nucl. Acids Res. (1992)20:533-538; Saison-Behmoaras et al., EMBO J. (1991) 10:1111-1118;Kabanov et al., FEBS Lett. (1990) 259: 327-330; Svinarchuk et al.,Biochimie (1993) 75:49-54; Manoharan et al., Tetrahedron Lett. (1995)36, 3651-3654; Shea et al., Nucl. Acids Res. (1990) 18:3777-3783;Manoharan et al., Nucleosides & Nucleotides (1995) 14: 969-973;Manoharan et al., Tetrahedron Lett. (1995) 36:3651-3654; Mishra et al.,Biochim. Biophys. Acta (1995) 1264:229-237; Crooke et al., J. Pharmacol.Exp. Ther. (1996) 277:923-937; U.S. Pat. No. 6,242,590.

Further included in the present invention is a method of high throughputscreening of antisense nucleic acids and modified versions thereof forbinding to targeted GENSET polynucleotide sequences or fragments thereof(see, e.g., U.S. Pat. No. 6,022,691).

The appropriate level of antisense nucleic acids required to inhibitgene expression may be determined using in vitro expression analysis.Typically, antisense molecules are introduced onto cell samples at anumber of different concentrations preferably between 1×10⁻¹⁰M to1×10⁻⁴M. Once the minimum concentration that can adequately control geneexpression is identified, the optimized dose is translated into a dosagesuitable for use in vivo or ex vivo. For example, an inhibitingconcentration in culture of 1×10⁻⁷ translates into a dose ofapproximately 0.6 mg/kg bodyweight.

An alternative to the antisense technology that is used according to thepresent invention comprises using ribozymes that will bind to a targetsequence via their complementary polynucleotide tail and that willcleave the corresponding RNA by hydrolyzing its target site (see, e.g.,Rossi et al., (1991) Pharmacol. Ther. 50:245-254 and Sczakiel et al.(1995)).

Triple Helix Approach

The GENSET genomic DNA may also be used to inhibit the expression of theGENSET gene based on intracellular triple helix formation (see, e.g.,Griffin et al., (1989) Science 245:967-971). To carry out gene therapystrategies using the triple helix approach, the sequences of the GENSETgenomic DNA are typically first scanned to identify 10-mer to 20-merhomopyrimidine or homopurine stretches which could be used intriple-helix based strategies for inhibiting GENSET gene expression.Following identification of candidate homopyrimidine or homopurinestretches, their efficiency in inhibiting GENSET gene expression isassessed by introducing varying amounts of oligonucleotides containingthe candidate sequences into tissue culture cells which express theGENSET gene. The oligonucleotides can be introduced into the cells usinga variety of methods known to those skilled in the art, and treatedcells are monitored for altered cell function or reduced GENSET geneexpression.

The oligonucleotides which are effective in inhibiting gene expressionin tissue culture cells may then be introduced in vivo using thetechniques and at a dosage calculated based on the in vitro results, asdescribed in the section entitled “Antisense Approach”.

Treating GENSET Gene-Related Disorders

The present invention further relates to methods, uses of GENSETpolypeptides and polynucleotides, and uses of modulators of GENSETpolypeptides and polynucleotides, for treating diseases/disordersassociated with GENSET genes by increasing or decreasing GENSET geneactivity and/or expression. These methodologies can be effected usingcompounds selected using screening protocols such as those describedherein and/or by using the gene therapy and antisense approachesdescribed in the art and herein. Gene therapy can be used to effecttargeted expression of GENSET genes in any tissue, e.g. a tissueassociated with the disease or condition to be treated. The GENSETcoding sequence can be cloned into an appropriate expression vector andtargeted to a particular cell type(s) to achieve efficient, high levelexpression. Introduction of the GENSET coding sequence into target cellscan be achieved, for example, using particle mediated DNA delivery,(Haynes et al., (1996) J. Biotechnol. 44(1-3):37-42 and Maurer et al.,(1999) Mol Membr Biol. 16(1):129-40), direct injection of naked DNA(Levy et al., (1996) Gene Ther. 3(3):201-11; and Felgner (1996) Hum GeneTher. 7(15):1791-3), or viral vector mediated transport (Smith et al.,(1996) Antiviral Res. 32(2):99-115, Stone et al., (2000) J. Endocrinol.164(2):103-18; Wu and Ataai (2000), Curr Opin Biotechnol. 11(2):205-8).Tissue specific effects can be achieved, for example, in the case ofvirus mediated transport by using viral vectors that are tissuespecific, or by the use of promoters that are tissue specific. Forinstance, any tissue-specific promoter may be used to achieve specificexpression, for example albumin promoters (liver specific; Pinkert etal., 1987 Genes Dev. 1:268-277), lymphoid specific promoters (Calame etal., 1988 Adv. Immunol. 43:235-275), promoters of T-cell receptors(Winoto et al., 1989 EMBO J. 8:729-733) and immunoglobulins (Banerji etal., 1983 Cell 33:729-740; Queen and Baltimore 1983 Cell 33:741-748),neuron-specific promoters (e.g. the neurofilament promoter; Byrne etal., 1989 PNAS 86:5473-5477), pancreas-specific promoters (Edlunch etal., 1985 Science 230:912-916) or mammary gland-specific promoters (milkwhey promoter, U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264, 166). Developmentally-regulated promoters can alsobe used, such as the murine homeobox promoters (Kessel et al., 1990Science 249:374-379) or the alpha-fetoprotein promoter (Campes et al.,1989 Genes Dev. 3:537-546).

Combinatorial approaches can also be used to ensure that the GENSETcoding sequence is activated in the target tissue (Butt and Karathanasis(1995) Gene Expr. 4(6):319-36; Miller and Whelan, (1997) Hum Gene Ther.8(7):803-15). Antisense oligonucleotides complementary to GENSET mRNAcan be used to selectively diminish or ablate the expression of theprotein (Wagner, et al. (1996), Nat. Biotechnol. 14(7):840-4)), forexample, at sites of inflammation. More specifically, antisenseconstructs or antisense oligonucleotides can be used to inhibit theproduction of GENSET in high expressing cells, e.g., by transfectingtarget cells with an expression vector comprising a GENSET genesequence, or portion thereof, in an antisense orientation relative tothe direction of transcription, or by introducing antisenseoligonucleotides directly into target cells. The therapeuticmethodologies described herein are applicable to both human andnon-human mammals.

Pharmaceutical and Physiologically Acceptable Compositions

The present invention also relates to pharmaceutical or physiologicallyacceptable compositions comprising, as active agent, the polypeptides,nucleic acids or antibodies of the invention. The invention also relatesto compositions comprising, as active agent, compounds selected usingthe above-described screening protocols. Such compositions include theactive agent in combination with a pharmaceutical or physiologicallyacceptable carriers such as a physiologically acceptable salt, ester, orsalt of such esters. In the case of naked DNA, the “carrier” may be goldparticles. The amount of active agent in the composition can vary withthe agent, the patient and the effect sought. Likewise, the dosingregimen can vary depending on the composition and the disease/disorderto be treated.

Therefore, the invention related to methods for the production ofpharmaceutical composition comprising a method for selecting an activeagent, compound, substance or molecule using any of the screening methoddescribed herein and furthermore mixing the identified active agent,compound, substance or molecule with a physiologically acceptablecarrier.

The term “physiologically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.Physiologically acceptable base addition salts are formed with metals oramines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci. (1977) 66: 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a physiologically acceptable salt of an acid form of one of thecomponents of the compositions of the invention. These include organicor inorganic acid salts of the amines. Preferred acid salts are thehydrochlorides, acetates, salicylates, nitrates and phosphates. Othersuitable physiologically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, hydrochloric acid, hydrobromic acid, sulfuricacid or phosphoric acid; with organic carboxylic, sulfonic, sulfo orphospho acids or N-substituted sulfamic acids, for example acetic acid,propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleicacid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lacticacid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citricacid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,the 20 alpha-amino acids involved in the synthesis of proteins innature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Physiologically acceptable salts of compounds may alsobe prepared with a physiologically acceptable cation. Suitablephysiologically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.For oligonucleotides, preferred examples of physiologically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to:parenteral, intracranial, intraorbital, intracapsular, intraspinal,intracisternal, intrapulmonary, oral, intravenous, intramuscular,intraarterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, rectal, intradermal, intravascular. In addition tothe active ingredients, these pharmaceutical compositions may containsuitable physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of Remington's Pharmaceutical Sciences (Maack Publishing Co.Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing physiologically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as powders, tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient. Suitable excipients are carbohydrateor protein fillers, such as sugars, including lactose, sucrose,mannitol, or sorbitol; starch from corn, wheat, rice, potato, or otherplants; cellulose, such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gumsincluding arabic and tragacanth; and proteins such as gelatin andcollagen. If desired, disintegrating or solubilizing agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, alginicacid, or a salt thereof, such as sodium alginate.

Formulations suitable for pulmonary or respiratory delivery include drypowders, liquid solutions or suspensions suitable for nebulization, andpropellant formulations suitable for use in metered dose inhalers(MDI's). In typical embodiments, dry powder formulations will have aparticle size within a preferred range for deposition within thealveolar region of the lung, typically from 0.5 μm to 5 μm. Respirablepowders of pharmaceutical compositions within the preferred size rangecan be produced by a variety of conventional techniques, such asjet-milling, spray-drying, solvent precipitation, and the like. Drypowders can then be administered to the patient in conventional drypowder inhalers (DPI's) that use the patient's inspiratory breaththrough the device to disperse the powder or in air-assisted devicesthat use an external power source to disperse the powder into an aerosolcloud (see, e.g., U.S. Pat. No. 5,458,135).

Liquid formulations for use in nebulizer systems preferably employslightly acidic buffers (pH 4-6) such as acetate, ascorbate, andcitrate, at concentrations of 5 mM to 50 mM. These buffers can act asantioxidants. Physiologically acceptable components to enhance ormaintain chemical stability include: antioxidants, chelating agents,protease inhibitors, isotonic modifiers, inert gases, and the like. Apreferred type of nebulizer suitable for delivering such liquidformulations is described in U.S. Pat. No. 5,458,135.

For use in MDI's, the pharmaceutical composition will typically beprocessed into respirable particles as described for the dry powderformulations, and the particles then suspended in a suitable aerosolpropellant (such as a CFC or HFC), typically being coated with asurfactant to enhance their dispersion. Such aerosol propellantformulations may further include a lower alcohol, such as ethanol (up to30% by weight) and other additives to maintain or enhance chemicalstability and physiological acceptability (see, e.g., U.S. Pat. No.6,080,721).

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethylcellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

In one embodiment, the preparation is a lyophilized powder which maycontain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose,and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined withbuffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of a GENSET polypeptide, such labelingwould include, e.g., amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart. For any compound, a therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example a GENSET polypeptide or fragments thereof,antibodies specific to GENSET polypeptides, agonists, antagonists orinhibitors of GENSET polypeptides, which ameliorates the symptoms orcondition. Therapeutic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED50 (the dose therapeutically effective in 50% of thepopulation) and LD50 (the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, LD50/ED50. Pharmaceuticalcompositions which exhibit large therapeutic indices are preferred. Thedata obtained from cell culture assays and animal studies is used informulating a range of dosage for human use. The dosage contained insuch compositions is preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Other factors that maybe considered when evaluating the proper dosage include the chemicalnature of the compound destined for delivery, the biological responsesassociated with the compound (both intended and coincidental) andanticipated contraindications. Additionally, the mode of delivery, theduration and frequency of administration (e.g. n doses per hours, ndoses per day, n doses per week, cumulative dosage per day, cumulativedosage per week), the biologically effective dose delivered to targetsite, often indicated by plasma level concentrations, and the rate orefficiency of compound clearance from the body may be considered.Long-acting pharmaceutical compositions may be administered, e.g., every3 to 4 days, every week, or once every two weeks depending on half-lifeand clearance rate of the particular formulation.

Normal dosage amounts may vary depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc. In general, for a 75 kgindividual the normal dosage range are as follows: for a small moleculecompound an effective does is usually between 0.3-50 mg/kg; forrecombinant polypeptides an effective dose is usually between 0.25-7.5mg/kg; for compounds used for mediating humoral immune responses (e.g.,polyvalent pneumococcal vaccine, Rh_(o) (D) immune globulin, Hepatitis Bvaccine, anti-CD20 antigen) the effective dose is usually between0.0015-1.5 mg/kg; for hormone supplemental compounds (e.g. estradiol,norethindrone) the effective dose is usually between 0.0005-0.5 mg/kgdepending upon delivery system utilized (e.g. transdermal, oral,topical).

Transdermal delivery systems (e.g. estradiol transdermal system,transdermal scopolamine system, transfermal nicotine patch) must becalibrated for nominal delivery dosages based upon efficiency ofpercutaneous delivery for the individual and specific compounds, surfacearea (cm²) of transdermal system contact, quantity and form of compoundintegrated into transdermal delivery system and anatomical location ofpositioned transdermal system. The effective dosage range of compoundsadministered in this manner is usually between 0.005-0.5 mg/kg

Uses of GENSET Sequences Computer-Related Embodiments

It will be appreciated by those skilled in the art that the nucleic acidcodes of the invention and polypeptide codes of the invention can bestored, recorded, and manipulated on any medium which can be read andaccessed by a computer. As used herein, the words “recorded” and“stored” refer to a process for storing information on a computermedium. A skilled artisan can readily adopt any of the presently knownmethods for recording information on a computer readable medium togenerate manufactures comprising one or more of the nucleic acid codesof the invention, or one or more of the polypeptide codes of theinvention. Another aspect of the present invention is a computerreadable medium having recorded thereon at least 1, 2, 5, 10, 15, 20,25, 30, or 50 nucleic acid or polypeptide codes of the invention.

Computer readable media include magnetically readable media, opticallyreadable media, electronically readable media and magnetic/opticalmedia. For example, the computer readable media may be a hard disk, afloppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD),Random Access Memory (RAM), or Read Only Memory (ROM).

Embodiments of the present invention include systems, particularlycomputer systems which store and manipulate the sequence informationdescribed herein. As used herein, “a computer system” refers to thehardware components, software components, and data storage componentsused to analyze the nucleotide sequences of the nucleic acid codes ofthe invention or the amino acid sequences of the polypeptide codes ofthe invention. The computer system preferably includes a processor forprocessing, accessing and manipulating the sequence data. The processorcan be any well-known type of central processing unit, such as thePentium III from Intel Corporation, or similar processor from Sun,Motorola, Compaq or International Business Machines. Preferably, thecomputer system is a general purpose system that comprises the processorand one or more internal data storage components for storing data, andone or more data retrieving devices for retrieving the data stored onthe data storage components. A skilled artisan can readily appreciatethat any one of the currently available computer systems are suitable.

In some embodiments, the computer system may comprise a sequencecomparer for comparing the above-described nucleic acid codes of theinvention or the polypeptide codes of the invention stored on a computerreadable medium to reference nucleotide or polypeptide sequences storedon a computer readable medium or present in a database. A “sequencecomparer” refers to one or more programs which are implemented on thecomputer system to compare a nucleotide or polypeptide sequence withother nucleotide or polypeptide sequences and/or compounds including butnot limited to peptides, peptidomimetics, and chemicals stored withinthe data storage means. For example, the sequence comparer may comparethe nucleotide sequences of nucleic acid codes of the invention or theamino acid sequences of the polypeptide codes of the invention stored ona computer readable medium to reference sequences stored on a computerreadable medium to identify homologies, motifs implicated in biologicalfunction, or structural motifs. The various sequence comparer programsidentified elsewhere in this patent specification are particularlycontemplated for use in this aspect of the invention. Alternatively, anucleotide or amino acid sequence is compared with a database ofsequences in order to determine the homology levels between the sequenceand the sequences in the database. The database of sequences can be aprivate database stored within the computer system, or a public databasesuch as GENBANK, PIR OR SWISSPROT that is available, e.g., through theInternet.

Accordingly, one aspect of the present invention is a computer systemcomprising a processor, a data storage device having stored thereon anucleic acid code of the invention or a polypeptide code of theinvention, a data storage device having retrievably stored thereonreference nucleotide sequences or polypeptide sequences to be comparedto the nucleic acid code of the invention or polypeptide code of theinvention and a sequence comparer for conducting the comparison.

Alternatively, the computer program may be a computer program whichcompares the nucleotide sequences of the nucleic acid codes of thepresent invention, to reference nucleotide sequences in order todetermine whether the nucleic acid code of the invention differs from areference nucleic acid sequence at one or more positions. Optionallysuch a program records the length and identity of inserted, deleted orsubstituted nucleotides with respect to the sequence of either thereference polynucleotide or the nucleic acid code of the invention. Inone embodiment, the computer program may be a program which determineswhether the nucleotide sequences of the nucleic acid codes of theinvention contain one or more single nucleotide polymorphisms (SNP) withrespect to a reference nucleotide sequence. These single nucleotidepolymorphisms may each comprise a single base substitution, insertion,or deletion. The method may also be performed by reading at least 2, 5,10, 15, 20, 25, 30, or 50 of the nucleic acid codes of the invention andthe reference nucleotide sequences through the use of the computerprogram and identifying differences between the nucleic acid codes andthe reference nucleotide sequences with the computer program.

In other embodiments the computer based system may further comprise anidentifier for identifying features within the nucleotide sequences ofthe nucleic acid codes of the invention or the amino acid sequences ofthe polypeptide codes of the invention. An “identifier” refers to one ormore programs which identifies certain features within theabove-described nucleotide sequences of the nucleic acid codes of theinvention or the amino acid sequences of the polypeptide codes of theinvention. In one embodiment, the identifier may comprise a programwhich identifies an open reading frame in the cDNAs codes of theinvention.

In additional embodiments, the present amino acid sequences can beanalyzed using any of a number of computer programs to identifystructural features of the protein, such as secondary, tertiary, andquarternary structures, to identify potential binding partners, etc. Theresults of the molecular modeling analysis may then be used, e.g., inrational drug design techniques to identify agents which modulate theactivity of the polypeptide codes of the invention.

Accordingly, another aspect of the present invention is a method ofidentifying a feature within the nucleic acid codes of the invention orthe polypeptide codes of the invention comprising reading the nucleicacid code(s) or the polypeptide code(s) through the use of a computerprogram which identifies features therein and identifying featureswithin the nucleic acid code(s) or polypeptide code(s) with the computerprogram. The method may be performed by reading a single sequence or atleast 2, 5, 10, 15, 20, 25, 30, or 50 of the nucleic acid codes of theinvention or the polypeptide codes of the invention through the use ofthe computer program and identifying features within the nucleic acidcodes or polypeptide codes with the computer program.

Motifs which may be detected using the above programs include leucinezippers, helix-turn-helix motifs, glycosylation sites, ubiquitinationsites, alpha helices, beta sheets, signal sequences, sequencesimplicated in transcription regulation such as homeoboxes, acidicstretches, enzymatic active sites, substrate binding sites, andenzymatic cleavage sites.

Although this invention has been described in terms of certain preferredembodiments, other embodiments which will be apparent to those ofordinary skill in the art in view of the disclosure herein are alsowithin the scope of this invention. Accordingly, the scope of theinvention is intended to be defined only by reference to the appendedclaims.

Throughout this application, various publications, kit manuals, patentsand published patent applications are cited. The entire disclosures ofeach of these publications, patents, manuals and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains. TABLE I SEQ ID Sequence ATCC ATCC NO. Type CloneID_Clone Name Name Deposit Deposit Date 1 DNA 243525_116-119-1-0-G6-FPCNAlt 2 Protein 243525_116-119-1-0-G6-F PCNAlt 3 DNA643144_181-17-2-0-A12-F Lectir 4 Protein 643144_181-17-2-0-A12-F Lectir5 DNA 212950.cREC_116-075-2-0- vlADAM20 H1-F 6 Protein212950.cREC_116-075-2-0- vlADAM20 H1-F 7 DNA 1000849866_181-44-3-0-Lipoglobulin A9-F 8 Protein 1000849866_181-44-3-0- Lipoglobulin A9-F 9DNA 164341_117-001-5-0-E2-F PBK 10 Protein 164341_117-001-5-0-E2-F PBK11 DNA 1000837037_228-43-2-0- Myeloidin C3-F 12 Protein1000837037_228-43-2-0- Myeloidin C3-F 13 DNA 101005_105-020-4-0-H11-FvACT 14 Protein 101005_105-020-4-0-H11-F vACT 15 DNA500743419_188-281-3-0- Claudinyn-5 H5-F 16 Protein500743419_188-281-3-0- Claudinyn-5 H5-F 17 DNA 645730_181-16-1-0-G9-FBenzodiazepine receptor 3 (BZRP-R3) 18 Protein 645730_181-16-1-0-G9-FBenzodiazepine receptor 3 (BZRP-R3) 19 DNA 646762_181-21-2-0-A3-FBenzodiazepine receptor 4 (BZRP-R4) 20 Protein 646762_181-21-2-0-A3-FBenzodiazepine receptor 4 (BZRP-R4) 21 DNA 420594_145-19-4-0-E7-FScolakin 22 Protein 420594_145-19-4-0-E7-F Scolakin 23 DNA119658_105-067-2-0-H4-F DOV 24 Protein 119658_105-067-2-0-H4-F DOV 25DNA 500706437_204-5-2-0-C9-F Placentalin 26 Protein500706437_204-5-2-0-C9-F Placentalin 27 DNA 176355_117-005-2-0-H11-FNAAR 28 Protein 176355_117-005-2-0-H11-F NAAR 29 DNA222588_116-094-1-0-H2-F Neurexinal 30 Protein 222588_116-094-1-0-H2-FNeurexinal 31 DNA 119033_105-066-4-0-F10-F NPAISY 32 Protein119033_105-066-4-0-F10-F NPAISY 33 DNA 125402_105-074-4-0-F3-F vCRTL-134 Protein 125402_105-074-4-0-F3-F vCRTL-1 35 DNA 107640_105-036-2-0-H3LIRION 36 Protein 107640_105-036-2-0-H3 LIRION 37 DNA588394_160-105-40-A11-F SLAMP 38 Protein 588394_160-105-40-A11-F SLAMP39 DNA 495718_160-26-2-0-E12-F SAP-MU-10 40 Protein495718_160-26-2-0-E12-F SAP-MU-10 41 DNA 612386_187-9-4-0-B2-F Cytogram42 Protein 612386_187-9-4-0-B2-F Cytogram 43 DNA 1000838982_220-20-4-0-Tetranab B7-F 44 Protein 1000838982_220-20-4-0- Tetranab B7-F 45 DNA500720840_205-20-1-0-B7-F PDI 46 Protein 500720840_205-20-1-0-B7-F PDI47 DNA 146821_106-020-1-0-G3-F NBART 48 Protein 146821_106-020-1-0-G3-FNBART 49 DNA 644724_181-21-1-0-A12-F NBTG 50 Protein644724_181-21-1-0-A12-F NBTG 51 DNA 583702_181-8-4-0-C8-F vITH1 52Protein 583702_181-8-4-0-C8-F vITH1

TABLE II Signal Mature Polyadenylation SEQ ID NO: ORF Peptide peptideSignal PolyA tail 1 [225-593] — — [769-774] [800-850] 3  [91-777] — —[826-831] [848-883] 5  [235-2562] — — — — 7 [264-926] [264-320][321-926] [1404-1409] [1421-1436] 9  [93-551]  [93-197] [198-551][2085-2090] [2117-2132] 11 [201-986] [201-269] [270-986] [2233-2238][2251-2266] 13  [66-1334]  [66-134]  [135-1334] [1558-1563] [1582-1597]15 [153-806] — — [1362-1367] [1382-1397] 17  [63-572] — — [750-755][767-782] 19  [63-572] — — [750-755] [774-789] 21  [86-403] — —[504-509] [540-555] 23  [415-1653] — — — [1688-1726] 25  [76-339] [76-147] [148-339] [903-908] [926-941] 27  [21-1118] [21-89]  [90-1118][1858-1863] [1879-1894] 29  [32-559] — — [702-707] [728-742] 31  [4-1533] — — [1709-1714] [1744-1766] 33  [11-802] [11-64]  [65-802][836-841] [862-877] 35  [38-1378]  [38-106]  [107-1378]  [107-1378] — 37 [330-1478] [330-398]  [399-1478] [1722-1727] [1742-1757] 39  [81-1517]— — [2786-2791] [2804-2818] 41 [121-546] [121-231] [232-546] [739-744][755-770] 43 [136-501] — — [1232-1237] [1255-1340] 45  [118-1632][118-189]  [190-1632] [1937-1942] [1956-1999] 47 [154-546] — — —[722-836] 49 [196-708] — — — [847-862] 51  [62-778]  [62-124] [125-778][912-917] [932-937]

TABLE III SEQ ID NO: Positions of immunogenic epitopes 4 1 . . . 8:12 .. . 17:57 . . . 64:63 . . . 74:90 . . . 114:122 . . . 132:162 . . .174:186 . . . 194:196 . . . 206:207 . . . 214 6 3 . . . 19:20 . . .28:79 . . . 89:100 . . . 107:150 . . . 164:209 . . . 221:333 . . .343:368 . . . 373:398 . . . 406:418 . . . 432: 430 . . . 439:447 . . .453:513 . . . 521:524 . . . 539:553 . . . 565:569 . . . 583:582 . . .593: 635 . . . 648:684 . . . 692:694 . . . 733:770 . . . 775 8 22 . . .31:39 . . . 45:81 . . . 90:94 . . . 99:98 . . . 106:108 . . . 116:136 .. . 144:183 . . . 191: 190 . . . 196:205 . . . 217 10 11 . . . 21:50 . .. 60:77 . . . 84:118 . . . 126 12 81 . . . 88:142 . . . 150:180 . . .186:194 . . . 203:219 . . . 227:251 . . . 261 14 25 . . . 31:34 . . .45:124 . . . 133:182 . . . 193:216 . . . 221:219 . . . 230:230 . . .239:267 . . . 272:279 . . . 285: 296 . . . 302:317 . . . 325:416 . . .422 16 58 . . . 63:188 . . . 194:199 . . . 217 18 22 . . . 36:151 . . .156:161 . . . 169 20 22 . . . 28:31 . . . 36:151 . . . 156:161 . . . 16922 21 . . . 31:71 . . . 77:77 . . . 105 24 1 . . . 14:20 . . . 44:50 . .. 59:82 . . . 99:101 . . . 113:153 . . . 162:163 . . . 169:175 . . .194:254 . . . 259:291 . . . 298: 326 . . . 331:353 . . . 361:368 . . .378:381 . . . 386:394 . . . 406 26 24 . . . 33:52 . . . 62:67 . . . 7828 21 . . . 42:79 . . . 91:122 . . . 132:182 . . . 189:264 . . . 277:286. . . 293:314 . . . 321:326 . . . 336:354 . . . 363 30 1 . . . 17:22 . .. 28:30 . . . 42:48 . . . 55:93 . . . 101 32 28 . . . 35:63 . . . 75:72. . . 82:87 . . . 95:152 . . . 162:176 . . . 185:196 . . . 212:224 . . .243:262 . . . 270:355 . . . 363: 369 . . . 374:407 . . . 417:455 . . .462:471 . . . 480:489 . . . 503 34 50 . . . 59:77 . . . 82:88 . . .98:108 . . . 120:208 . . . 216:234 . . . 241 36 16 . . . 22:63 . . .83:87 . . . 112:145 . . . 151:168 . . . 174:189 . . . 195:221 . . .232:236 . . . 244:281 . . . 291: 290 . . . 307:304 . . . 320:334 . . .341:344 . . . 355:361 . . . 373:418 . . . 433 38 29 . . . 39:39 . . .44:51 . . . 59:71 . . . 79:99 . . . 108:111 . . . 120:139 . . . 145:152. . . 157:164 . . . 173:185 . . . 195: 217 . . . 232:244 . . . 257:264 .. . 270:277 . . . 286:331 . . . 341:352 . . . 365:371 . . . 381 40 24 .. . 29:34 . . . 41:52 . . . 58:92 . . . 104:124 . . . 131:180 . . .198:332 . . . 340:403 . . . 411:422 . . . 433:446 . . . 455 42 66 . . .73:91 . . . 102:113 . . . 130 46 31 . . . 41:38 . . . 43:58 . . . 63:86. . . 98:107 . . . 122:128 . . . 133:176 . . . 186:193 . . . 202:209 . .. 215:266 . . . 283: 341 . . . 349:354 . . . 368:368 . . . 378:390 . . .397:406 . . . 419:421 . . . 427:440 . . . 447:456 . . . 471:480 . . .485 48 98 . . . 105:123 . . . 130 50 30 . . . 40:53 . . . 68:114 . . .119:128 . . . 139:157 . . . 166 52 15 . . . 31:46 . . . 58:107 . . .113:150 . . . 155:172 . . . 184:197 . . . 210

1. An isolated polynucleotide, comprising a nucleic acid sequenceselected from the group consisting of: a) a polynucleotide of an odd SEQID NO., or of a human cDNA of a deposited clone, encoding at least anysingle integer from 6 to 776 amino acids of any one even SEQ ID NO.; b)a polynucleotide of an odd SEQ ID NO., or of a human cDNA of a depositedclone, encoding the signal peptide sequence of any one even SEQ ID NO.;c) a polynucleotide of an odd SEQ ID NO., or of a human cDNA of adeposited clone, encoding a mature polypeptide sequence of any one evenSEQ ID NO.; d) a polynucleotide of an odd SEQ ID NO., or of a human cDNAof a deposited clone, encoding a full length polypeptide sequence of anyone even SEQ ID NO.; e) a polynucleotide of an odd SEQ ID NO., or of ahuman cDNA of a deposited clone, encoding a polypeptide sequence of abiologically active fragment of any one even SEQ ID NO.; f) apolynucleotide encoding a polypeptide sequence of at least any singleinteger from 6 to 776 amino acids of any one even SEQ ID NO. or of apolypeptide encoded by a human cDNA of a deposited clone; g) apolynucleotide encoding a polypeptide sequence of a signal peptide ofany one even SEQ ID NO. or of a signal peptide encoded by a human cDNAof a deposited clone; h) a polynucleotide encoding a polypeptidesequence of a mature polypeptide of any one even SEQ ID NO. or of amature polypeptide encoded by a human cDNA of a deposited clone; i) apolynucleotide encoding a polypeptide sequence of a full lengthpolypeptide of any one even SEQ ID NO. or of a mature polypeptideencoded by a human cDNA of a deposited clone; j) a polynucleotideencoding a polypeptide sequence of a biologically polypeptide of any oneeven SEQ ID NO., or of a biologically polypeptide encoded by a humancDNA of a deposited clone; k) a polynucleotide of any one of a) throughj) further comprising an expression vector; l) a host cell recombinantfor a polynucleotide of a) through k) above; m) a non-human transgenicanimal comprising the host cell of k); or n) a polynucleotide of a)through j) further comprising a physiologically acceptable carrier.
 2. Apolypeptide comprising an amino acid sequence selected from the groupconsisting of: a) any single integer from 6 to 776 amino acids of anyone of SEQ ID NO. 2, 4, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or 52 or of a polypeptide asencoded by a human cDNA of a deposited clone; b) a signal peptidesequence of any one of SEQ ID NO. 2, 4, 6, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or 52 or asencoded by a human cDNA of a deposited clone; c) a mature polypeptidesequence of any one of SEQ ID NO. 2, 4, 6, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or 52 or asencoded by a human cDNA of a deposited clone; d) a full lengthpolypeptide sequence of any one of SEQ ID NO. 2, 4, 6, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or 52or as encoded by a human cDNA of a deposited clone; or e) a polypeptideof a) through d) further comprising a physiologically acceptablecarrier.
 3. A method of making a polypeptide, said method comprising a)providing a population of host cells comprising the polynucleotide ofclaim 1; b) culturing said population of host cells under conditionsconducive to the production of a polypeptide within said host cells; andc) purifying said polypeptide from said population of host cells.
 4. Amethod of making a polypeptide, said method comprising: a) providing apopulation of cells comprising a polynucleotide encoding a polypeptideof SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or 52, operably linked to apromoter; b) culturing said population of cells under conditionsconducive to the production of said polypeptide within said cells; andc) purifying said polypeptide from said population of cells.
 5. Anantibody that specifically binds to the polypeptide comprising SEQ IDNO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50 or
 52. 6. A method of binding a polypeptideto an antibody, comprising contacting antibody according to claim 5 withsaid polypeptide under conditions in which antibody can specificallybind to said polypeptide.
 7. A method of determining whether a GENSETgene is expressed within a mammal, said method comprising the steps of:a) providing a biological sample from said mammal; b) contacting saidbiological sample with either of: i) a polynucleotide that hybridizesunder stringent conditions to the polynucleotide of claim 1; or ii) anantibody according to claim 5; and c) detecting the presence or absenceof hybridization between said polynucleotide and an RNA species withinsaid sample, or the presence or absence of binding of said polypeptideto a protein within said sample; wherein a detection of saidhybridization or of said binding indicates that said GENSET gene isexpressed within said mammal.
 8. The method of claim 7, wherein saidpolynucleotide is a primer, and wherein said hybridization is detectedby detecting the presence of an amplification product comprising thesequence of said primer.
 9. The method of claim 7, wherein said antibodyis monoclonal.
 10. A method of determining whether a mammal has anelevated or reduced level of GENSET gene expression, said methodcomprising the steps of: a) providing a biological sample from saidmammal; and b) comparing the amount of SEQ ID NO. 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50 or 52, or of an RNA species encoding said polypeptide, within saidbiological sample with a level detected in or expected from a controlsample; wherein an increased amount of said polypeptide or said RNAspecies within said biological sample compared to said level detected inor expected from said control sample indicates that said mammal has anelevated level of said GENSET gene expression, and wherein a decreasedamount of said polypeptide or said RNA species within said biologicalsample compared to said level detected in or expected from said controlsample indicates that said mammal has a reduced level of said GENSETgene expression.
 11. A method of identifying a candidate modulator of aGENSET polypeptide, said method comprising: a) contacting thepolypeptide comprising SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or 52 with atest compound; and b) determining whether said compound specificallybinds to said polypeptide; wherein a detection that said compoundspecifically binds to said polypeptide indicates that said compound is acandidate modulator of said GENSET polypeptide.
 12. The method of claim11, further comprising testing the biological activity of said GENSETpolypeptide in the presence of said candidate modulator, wherein analteration in the biological activity of said GENSET polypeptide in thepresence of said compound in comparison to the activity in the absenceof said compound indicates that the compound is a modulator of saidGENSET polypeptide.
 13. A method for the production of a pharmaceuticalcomposition comprising: a) identifying a modulator of a GENSETpolypeptide using the method of claim 11; and b) combining saidmodulator with a physiologically acceptable carrier.